本申请是申请日为2005年11月14日、申请号为201310125428.4、发明名称为“FVIII的位点定向修饰”的PCT申请的分案申请。This application is a divisional application of a PCT application with a filing date of November 14, 2005, an application number of 201310125428.4, and an invention title of "site-directed modification of FVIII".
与相关申请的交叉参考Cross references to related applications
本申请要求享有2004年11月12日提交的美国专利申请系列号60/627,277的优先权利益，其在这里整体引作参考。This application claims the benefit of priority to US Patent Application Serial No. 60/627,277, filed November 12, 2004, which is hereby incorporated by reference in its entirety.
技术领域technical field
本发明涉及因子VIII(FVIII)突变蛋白，其允许在确定的位点偶联一个或多个生物相容的聚合物如聚乙二醇。另外，提供了用于治疗目的的相关的制剂、其剂量及施用方法。这些修饰的FVIII变体和相关的组合物和方法用于给遭受血友病A的个体提供具有减少的注射频率和减少的免疫原性应答的治疗选择。The present invention relates to Factor VIII (FVIII) muteins which allow conjugation at defined sites to one or more biocompatible polymers such as polyethylene glycol. In addition, relevant formulations, dosages and methods of administration thereof for therapeutic purposes are provided. These modified FVIII variants and related compositions and methods are useful in providing treatment options with reduced injection frequency and reduced immunogenic responses to individuals suffering from hemophilia A.
背景技术Background technique
血友病A是最常见的遗传性凝结障碍，估计的发病率为1/5000男性。它的原因是FVIII的缺乏或结构缺陷，所述FVIII是血液凝结的固有途径中的关键组分。目前血友病A的治疗包含静脉内注射人FVIII。已经重组地生产了人FVIII，作为约300kD的单链分子。它由结构域A1-A2-B-A3-C1-C2组成(Thompson，2003，Semin.Hematol.29，pp.11-22)。前体产物在高尔基体中被加工成200kD(重链)和80kD(轻链)的2条多肽链，这2条链通过金属离子结合到一起(Kaufman等，1988，J.Biol.Chem.263，p.6352；Andersson等，1986，Proc.Natl.Acad.Sci.83，p.2979)。Hemophilia A is the most common inherited clotting disorder, with an estimated prevalence of 1 in 5000 males. It is caused by a deficiency or structural defect of FVIII, a key component in the intrinsic pathway of blood coagulation. Current treatment for hemophilia A consists of intravenous injection of human FVIII. Human FVIII has been produced recombinantly as a single-chain molecule of approximately 300 kD. It consists of domains A1-A2-B-A3-C1-C2 (Thompson, 2003, Semin. Hematol. 29, pp. 11-22). The precursor product is processed into 2 polypeptide chains of 200kD (heavy chain) and 80kD (light chain) in the Golgi apparatus, and these 2 chains are bound together by metal ions (Kaufman et al., 1988, J.Biol.Chem.263 , p.6352; Andersson et al., 1986, Proc. Natl. Acad. Sci. 83, p.2979).
FVIII的B-结构域似乎是可有可无的，因为还已经显示B-结构域缺失的FVIII(BDD，90kD A1-A2重链+80kD轻链)可以有效地用作血友病A的替补疗法。B-结构域缺失的FVIII序列含有B-结构域的14个氨基酸以外的所有缺失。The B-domain of FVIII appears to be dispensable, as it has also been shown that B-domain deleted FVIII (BDD, 90kD A1-A2 heavy chain + 80kD light chain) can be effectively used as a replacement for hemophilia A therapy. The B-domain deleted FVIII sequence contains all but 14 amino acids of the B-domain deleted.
目前，通过根据需要静脉内施用FVIII来治疗血友病A患者，或者作为预防疗法每周施用几次。关于预防治疗，每周施用15-25IU/kg体重的因子VIII 3次。患者总是需要它。因为它在人中的半衰期短，所以必须频繁地施用FVIII。尽管全长蛋白具有大于300kD的大尺寸，但FVIII具有仅约11小时的人中的半衰期(Ewenstein等，2004，Semin.Hematol.41，pp.1-16)。对频繁的静脉内注射的需要造成患者顺应性的巨大障碍。如果可以开发具有更长的半衰期且因而需要更低频率的施用的FVIII产物，则会更方便患者。另外，如果提高了半衰期，那么由于需要更低的剂量，可以降低治疗费用。Currently, hemophilia A patients are treated by administering FVIII intravenously as needed, or several times a week as prophylactic therapy. For prophylactic treatment, factor VIII is administered 15-25 IU/kg body weight 3 times a week. Patients need it all the time. Because of its short half-life in humans, FVIII must be administered frequently. Despite the large size of the full-length protein, greater than 300 kD, FVIII has a half-life in humans of only about 11 hours (Ewenstein et al., 2004, Semin. Hematol. 41, pp. 1-16). The need for frequent intravenous injections poses a significant barrier to patient compliance. It would be more convenient for patients if a FVIII product could be developed that had a longer half-life and thus required less frequent administration. In addition, if the half-life is increased, the cost of treatment can be reduced since lower doses are required.
现有疗法的另一个缺点是，约25-30％患者发展抑制FVIII活性的抗体(Saenko等，2002，Haemophilia 8，pp.1-11)。抑制性抗体的主要表位定位于A2结构域中的残基484-508、A3结构域中的残基1811-1818和C2结构域。抗体发展阻止FVIII作为替补疗法的应用，迫使该组患者寻求使用高剂量重组因子VIIa的甚至更昂贵的治疗和免疫耐受疗法。Another disadvantage of existing therapies is that approximately 25-30% of patients develop antibodies that inhibit FVIII activity (Saenko et al., 2002, Haemophilia 8, pp. 1-11). The major epitopes of the inhibitory antibodies were localized to residues 484-508 in the A2 domain, residues 1811-1818 in the A3 domain and the C2 domain. Antibody development prevents the use of FVIII as an alternative therapy, forcing this group of patients to seek even more expensive treatment with high doses of recombinant Factor Vila and immune tolerance therapy.
下面的研究鉴别出了抑制性抗体的FVIII表位。在25份抑制性血浆样品的研究中，发现11份结合凝血酶产生的73kD轻链片段A3C1C2，4份结合A2结构域，且10份结合二者(Fulcher，C.等，1985，Proc.Natl.Acad.Sci.2(22)，pp.7728-32)。在另一项研究中，重组A2多肽中和了8种来自患者的A2结构域抑制剂中的6种(Scandella，D.等，1993，Blood 82(6)，pp.1767-75)。将9种来自患者的抑制剂中的6种的表位作图到A2残基379-538(Scandella，D.等，1988，Proc.Natl.Acad.Sci.85(16)，pp.6152-6)。18种重链抑制剂的表位定位于相同的A2结构域N-末端18.3kD区域(Scandella，D.等，1989，Blood 74(5)，PP.1618-26)。The following studies identified FVIII epitopes for inhibitory antibodies. In a study of 25 inhibitory plasma samples, 11 were found to bind the thrombin-generated 73kD light chain fragment A3C1C2, 4 to the A2 domain, and 10 to both (Fulcher, C. et al., 1985, Proc. Natl .Acad.Sci.2(22), pp.7728-32). In another study, recombinant A2 polypeptides neutralized 6 of 8 patient-derived A2 domain inhibitors (Scandella, D. et al., 1993, Blood 82(6), pp. 1767-75). The epitope of 6 of 9 inhibitors from patients was mapped to A2 residues 379-538 (Scandella, D. et al., 1988, Proc. Natl. Acad. Sci. 85(16), pp. 6152- 6). The epitopes of 18 heavy chain inhibitors were mapped to the same N-terminal 18.3 kD region of the A2 domain (Scandella, D. et al., 1989, Blood 74(5), PP. 1618-26).
通过用同源猪序列替代人A2结构域残基387-604产生的有活性的重组杂种人/猪FVIII分子，对患者A2抑制剂是抗性的(Lubin，I.等，1994，J.Biol.Chem.269(12)，pp.8639-41)，且对与患者A2抑制剂竞争结合A2的鼠单克隆抗体mAB 413IgG是抗性的(Scandella，D.等，1992，Thromb Haemost.67(6)，pp.665-71)。当实验表明mAB 413IgG和4种患者抑制剂不抑制其中用猪序列替代A2结构域残基484-508的杂种人/猪FVIII时，该A2结构域表位进一步定位于A2结构域残基484-508(Healey，J.等，1995，J.Biol.Chem.270(24)，pp.14505-9)。该杂种FVIII还对筛选的23份患者血浆的至少一半是更高抗性的(Barrow，R.等，2000，Blood 95(2)，pp.564-8)。丙氨酸扫描诱变鉴别的残基487对结合测试的所有5种患者抑制剂都是关键的，而残基484、487、489和492对于与mAB 413 IgG的相互作用都是重要的(Lubin，I-，J.Biol.Chem.272(48)，pp.30191-5)。在接受R484A/R489A/P492A突变体(而不是R484A/R489A突变体)的小鼠中的抑制性抗体效价，显著低于接受对照人BDD FVIII的小鼠(Parker，E.等，2004，Blood 104(3)，pp.704-10)。总之，A2结构域的484-508区域似乎是FVIII活性的抑制剂的结合位点。Active recombinant hybrid human/pig FVIII molecules produced by substituting residues 387-604 of the human A2 domain with homologous porcine sequences are resistant to patient A2 inhibitors (Lubin, I. et al., 1994, J. Biol .Chem.269(12), pp.8639-41), and is resistant to the murine monoclonal antibody mAB 413 IgG which competes with the patient's A2 inhibitor for binding to A2 (Scandella, D. et al., 1992, Thromb Haemost.67( 6), pp. 665-71). This A2 domain epitope was further localized at A2 domain residues 484-508 when experiments showed that mAB 413 IgG and 4 patient inhibitors did not inhibit hybrid human/pig FVIII in which A2 domain residues 484-508 were replaced with porcine sequences 508 (Healey, J. et al., 1995, J. Biol. Chem. 270(24), pp. 14505-9). This hybrid FVIII was also more resistant to at least half of the 23 patient plasmas screened (Barrow, R. et al., 2000, Blood 95(2), pp.564-8). Alanine scanning mutagenesis identified residue 487 as critical for binding to all 5 patient inhibitors tested, whereas residues 484, 487, 489 and 492 were all important for interaction with mAB 413 IgG (Lubin , I-, J.Biol.Chem.272(48), pp.30191-5). Inhibitory antibody titers in mice receiving the R484A/R489A/P492A mutant (but not the R484A/R489A mutant) were significantly lower than mice receiving the control human BDD FVIII (Parker, E. et al., 2004, Blood 104(3), pp. 704-10). Taken together, the region 484-508 of the A2 domain appears to be the binding site for inhibitors of FVIII activity.
除了发展对FVIII的免疫应答以外，常规疗法的另一个问题是，其需要频繁的给药，因为FVIII的体内半衰期短。已经研究了从循环清除FVIII的机理。In addition to developing an immune response to FVIII, another problem with conventional therapy is that it requires frequent dosing due to the short in vivo half-life of FVIII. The mechanism of FVIII clearance from circulation has been investigated.
从循环清除FVIII，已部分地归因于向低密度脂蛋白受体-相关蛋白(LRP)的特异性结合，所述LRP是具有广泛配体特异性的肝清除受体(Oldenburg等，2004，Haemophilia10 Suppl 4，pp.133-139)。最近，还表明低密度脂蛋白(LDL)受体在FVIII清除中起作用，如通过与LRP协同调节FVIII的血浆水平(Bovenschen等，2005，Blood 106，pp.906-910)。两种相互作用都被结合细胞表面硫酸肝素蛋白聚糖(HSPG)所促进。当LRP被封闭时，可以使在小鼠中的血浆半衰期延长3.3倍，或者当LRP和细胞表面HSPG都被封闭时，可以延长5.5倍(Sarafanov等，2001，J.Biol.Chem.276，pp.11970-11979)。推测HSPG将FVIII集中在细胞表面上，并将其呈递给LRP。FVIII上的LRP结合位点已经被定位在A2残基484-509(Saenko等，1999，J.Biol.Chem.274，pp.37685-37692)、A3残基1811-1818(Bovenschen等，2003，J.Biol.Chem.278，pp.9370-9377)和C2结构域中的表位(Lenting等，1999，J.Biol.Chem.274，pp.23734-23739)。Clearance of FVIII from circulation has been attributed in part to specific binding to low-density lipoprotein receptor-related protein (LRP), a hepatic clearance receptor with broad ligand specificity (Oldenburg et al., 2004, Haemophilia 10 Suppl 4, pp. 133-139). Recently, low density lipoprotein (LDL) receptors have also been shown to play a role in FVIII clearance, eg by cooperating with LRP to regulate FVIII plasma levels (Bovenschen et al., 2005, Blood 106, pp. 906-910). Both interactions are facilitated by binding to cell surface heparan sulfate proteoglycans (HSPGs). The plasma half-life in mice can be extended 3.3-fold when LRP is blocked, or 5.5-fold when both LRP and cell surface HSPG are blocked (Sarafanov et al., 2001, J.Biol.Chem.276, pp .11970-11979). It is speculated that HSPG concentrates FVIII on the cell surface and presents it to LRP. The LRP binding site on FVIII has been mapped at A2 residues 484-509 (Saenko et al., 1999, J.Biol.Chem.274, pp.37685-37692), A3 residues 1811-1818 (Bovenschen et al., 2003, J. Biol. Chem. 278, pp. 9370-9377) and epitopes in the C2 domain (Lenting et al., 1999, J. Biol. Chem. 274, pp. 23734-23739).
通过蛋白酶的作用，也可以将FVIII从循环中清除。为了理解该作用，人们必须理解FVIII参与血液凝结的机理。FVIII作为结合vWF的重链和轻链的异源二聚体而循环。VWF结合包含FVIII残基1649-1689(Foster等，1988，J.Biol.Chem.263，pp.5230-5234)和C1(Jacquemin等，2000，Blood 96，pp.958-965)和C2结构域(Spiegel，P.等，2004，J.Biol.Chem.279(51)，pp.53691-8)的部分。FVIII被凝血酶激活，该凝血酶切割残基372、740和1689之后的肽键，以产生A1、A2和A3-C1-C2结构域的异源三聚体(Pittman等，2001，Proc.Natl.Acad.Sci.276，pp.12434-12439)。激活后，FVIII从vWF解离，并通过结合磷脂，集中在血小板细胞表面。磷脂结合包含FVIII残基2199、2200、2251和2252(Gilbert等，2002，J.Biol.Chem.277，pp.6374-6381)。在这里，它通过与FVIII残基558-565(Fay等，1994，J.Biol.Chem.269，pp.20522-20527)和1811-1818(Lenting等，1996，J.Biol.Chem.271，pp.1935-1940)的相互作用，结合FIX，并通过与FVIII残基349-372(Nogami等，2004，J。Biol.Chem.279，pp.15763-15771)的相互作用，结合FX，并作为FX的FIX激活的辅因子，所述FX是内源性凝结途径的基本组分。激活的FVIII(FVIIIa)受到蛋白酶激活的蛋白C(APC)的部分灭活，这通过在FVIII残基336和562后面的切割来实现(Regan等，1996，J.Biol.Chem.271，pp.3982-3987)。但是，灭活的主要决定因素是A2结构域从A1和A3-C1-C2解离(Fay等，1991，J.Biol.Chem.266，pp.8957-8962)。FVIII can also be cleared from circulation by the action of proteases. In order to understand this role, one must understand the mechanism by which FVIII is involved in blood coagulation. FVIII circulates as a heterodimer of vWF-bound heavy and light chains. VWF binding contains FVIII residues 1649-1689 (Foster et al., 1988, J. Biol. Chem. 263, pp.5230-5234) and C1 (Jacquemin et al., 2000, Blood 96, pp.958-965) and C2 domains (Spiegel, P. et al., 2004, J. Biol. Chem. 279(51), pp.53691-8). FVIII is activated by thrombin, which cleaves the peptide bond after residues 372, 740, and 1689 to generate a heterotrimer of the A1, A2, and A3-C1-C2 domains (Pittman et al., 2001, Proc. Natl .Acad.Sci.276, pp.12434-12439). After activation, FVIII dissociates from vWF and concentrates on the surface of platelet cells by binding to phospholipids. Phospholipid binding involves FVIII residues 2199, 2200, 2251 and 2252 (Gilbert et al., 2002, J. Biol. Chem. 277, pp. 6374-6381). Here, it is linked to FVIII residues 558-565 (Fay et al., 1994, J.Biol.Chem.269, pp.20522-20527) and 1811-1818 (Lenting et al., 1996, J.Biol.Chem.271, pp.1935-1940), binds FIX, and through the interaction with FVIII residues 349-372 (Nogami et al., 2004, J. Biol. Chem.279, pp.15763-15771), binds FX, and Acts as a cofactor for FIX activation of FX, an essential component of the intrinsic coagulation pathway. Activated FVIII (FVIIIa) is partially inactivated by protease-activated protein C (APC) by cleavage behind FVIII residues 336 and 562 (Regan et al., 1996, J. Biol. Chem. 271, pp. 3982-3987). However, the main determinant of inactivation is the dissociation of the A2 domain from A1 and A3-C1-C2 (Fay et al., 1991, J. Biol. Chem. 266, pp. 8957-8962).
已经证实增加蛋白的体内半衰期的一种方法是PEG化(PEGylation)。PEG化是长链聚乙二醇(PEG)分子向蛋白或其它分子的共价附着。PEG可以是线性形式或分支形式，以产生具有不同特征的不同分子。除了增加肽或蛋白的半衰期以外，PEG化已经用于减少抗体发展，保护蛋白免受蛋白酶消化，和保持材料在肾滤液的外面(Harris等，2001，ClinicalPharmacokinetics 40，pp.539-51)。另外，PEG化也可以增加蛋白的总稳定性和溶解度。最后，PEG化蛋白的持续的血浆浓度，可以通过减少药物的低谷至顶点水平降低不利的副作用的程度，从而消除对在早期时间点引入超生理水平的蛋白的需要。One method that has been shown to increase the in vivo half-life of proteins is PEGylation. PEGylation is the covalent attachment of long-chain polyethylene glycol (PEG) molecules to proteins or other molecules. PEG can be in linear or branched form to produce different molecules with different characteristics. In addition to increasing the half-life of peptides or proteins, PEGylation has been used to reduce antibody development, protect proteins from protease digestion, and keep material out of kidney filtrate (Harris et al., 2001, Clinical Pharmacokinetics 40, pp. 539-51). In addition, PEGylation can also increase the overall stability and solubility of the protein. Finally, sustained plasma concentrations of PEGylated proteins may reduce the magnitude of adverse side effects by reducing trough-to-peak levels of the drug, thereby eliminating the need to introduce supraphysiological levels of protein at early time points.
通过用大聚合物(如PEG和葡聚糖)靶向伯胺(N-末端和赖氨酸)来随机修饰FVIII的尝试，已经取得不同程度的成功(WO94/15625、美国专利4970300、美国专利6048720)。在1994年专利申请(WO94/15625)中公开的最显著的提高，显示了4倍半衰期提高，但是代价是，在全长FVIII与50倍摩尔过量的PEG反应后，丧失2倍活性。WO2004/075923公开了通过随机修饰生成的FVIII和聚乙二醇的缀合物。在过去，已经批准随机地PEG化的蛋白如干扰素α(Kozlowski等，2001，BioDrugs 15，pp.419-429)用作治疗剂。Attempts to randomly modify FVIII by targeting primary amines (N-terminal and lysine) with large polymers such as PEG and dextran have met with varying degrees of success (WO94/15625, US Patent 4970300, US Patent 6048720). The most dramatic improvement, disclosed in the 1994 patent application (WO94/15625), showed a 4-fold increase in half-life, but at the expense of a 2-fold loss of activity following the reaction of full-length FVIII with a 50-fold molar excess of PEG. WO2004/075923 discloses conjugates of FVIII and polyethylene glycol produced by random modification. Randomly PEGylated proteins such as interferon alpha (Kozlowski et al., 2001, BioDrugs 15, pp. 419-429) have been approved as therapeutic agents in the past.
但是，对于异源二聚体的FVIII，该随机方案更容易出问题。FVIII具有数百个潜在的PEG化位点，包括158个赖氨酸，2个N-末端和多个组氨酸、丝氨酸、苏氨酸和酪氨酸，它们都可能被主要靶向伯胺的试剂PEG化。例如，显示PEG化的干扰素α-2b的主要位置异构体是组氨酸(Wang等，2000，Biochemistry 39，pp.10634-10640)。此外，全长FVIII的不均匀加工，可以导致原材料的混合物，这导致PEG化的产物的进一步复杂性。不控制FVIII上的PEG化位点的另一个缺点是，如果PEG附着在关键的活性位点处或附近，尤其是如果超过一个PEG或单个大PEG缀合到FVIII上，那么潜在的活性降低。因为随机的PEG化总是产生大量多重PEG化的产物，所以仅仅得到单-PEG化的产物的纯化会急剧降低总得率。最后，产物概况的巨大异质性使每批的一致合成和表征几乎不可能。因为良好生产需要一致的、充分表征的产品，所以产物异质性是商业化的屏障。鉴于所有这些原因，需要更特异性的PEG化FVIII的方法。However, for heterodimeric FVIII, this randomization scheme is more prone to problems. FVIII has hundreds of potential PEGylation sites, including 158 lysines, 2 N-termini and multiple histidines, serines, threonines and tyrosines, all of which may be primarily targeted to primary amines Reagents for PEGylation. For example, it was shown that the major positional isomer of PEGylated interferon alpha-2b is histidine (Wang et al., 2000, Biochemistry 39, pp. 10634-10640). In addition, uneven processing of full-length FVIII can lead to mixtures of starting materials, which lead to further complexity of PEGylated products. Another disadvantage of not controlling the PEGylation site on FVIII is that potential activity is reduced if PEG is attached at or near critical active sites, especially if more than one PEG or a single large PEG is conjugated to FVIII. Since random PEGylation always yields large amounts of multiple PEGylated product, purification to obtain only mono-PEGylated product drastically reduces the overall yield. Finally, the enormous heterogeneity of product profiles makes consistent synthesis and characterization of each batch nearly impossible. Because good manufacturing requires a consistent, well-characterized product, product heterogeneity is a barrier to commercialization. For all of these reasons, more specific methods of PEGylating FVIII are needed.
在近期的综述中(Kochendoerfer，G.，Curr.Opin.Chem.Biol.2005，可在线以Oct.15，2005，direct object identifierdoi：10.1016/j.cbpa.2005.10.007得到)，已经总结了各种位点定向蛋白PEG化策略。一种方法包含，通过化学合成或重组表达，将非天然氨基酸掺入蛋白，随后添加将与非天然氨基酸特异性地反应的PEG衍生物。例如，非天然氨基酸可以是含有不存在于天然蛋白中的酮基的氨基酸。但是，蛋白的化学合成对于如FVIII这样大的蛋白不可行。目前肽合成的界限是约50个残基。可以连接几个肽，以形成更大的多肽片，但是生产甚至B-结构域缺失的FVIII，会需要超过20次连接，这会导致小于1％回收，甚至在理想的反应条件下。迄今为止，含有非天然氨基酸的蛋白的重组表达主要限于非哺乳动物表达系统。对于需要在哺乳动物系统中表达的大且复杂的蛋白如FVIII，预期该方法有问题。In a recent review (Kochendoerfer, G., Curr. Opin. Chem. Biol. 2005, available online as Oct. 15, 2005, direct object identifierdoi: 10.1016/j.cbpa.2005.10.007 ), it has been summarized that Various site-directed protein PEGylation strategies. One approach involves the incorporation of an unnatural amino acid into a protein, by chemical synthesis or recombinant expression, followed by the addition of a PEG derivative that will specifically react with the unnatural amino acid. For example, an unnatural amino acid can be an amino acid that contains a keto group that is not found in natural proteins. However, chemical synthesis of proteins is not feasible for proteins as large as FVIII. The current limit for peptide synthesis is about 50 residues. Several peptides can be ligated to form larger polypeptide pieces, but producing even B-domain-deleted FVIII would require more than 20 ligations, which would result in less than 1% recovery, even under ideal reaction conditions. To date, recombinant expression of proteins containing unnatural amino acids has been largely limited to non-mammalian expression systems. This approach is expected to be problematic for large and complex proteins such as FVIII that need to be expressed in mammalian systems.
蛋白的位点特异性的PEG化的另一种方法是，用PEG-醛靶向N-末端主链胺。在该方法中需要低pH来实现超过其它胺基团的特异性。但是，这与FVIII的稳定性所需的狭窄的接近中性的pH范围不相容(Wang等，2003，International J.Pharmaceutics 259，pp.1-15)。此外，FVIII的N-末端PEG化不会导致提高的血浆半衰期，如果该区域不参与血浆清除的话。实际上，FVIII轻链的N-末端区域已经参与结合von Willebrand因子(vWF)，后者是对FVIII在循环中的存活至关重要的载体蛋白。通过因子VIII的N-末端修饰，可以破坏或减弱与vWF的至关重要的结合。因而，FVIII的N-末端PEG化可能具有降低FVIII的血浆半衰期的相反作用。Another approach to site-specific PEGylation of proteins is to target the N-terminal backbone amine with a PEG-aldehyde. Low pH is required in this method to achieve specificity over other amine groups. However, this is incompatible with the narrow near-neutral pH range required for the stability of FVIII (Wang et al., 2003, International J. Pharmaceutics 259, pp. 1-15). Furthermore, N-terminal PEGylation of FVIII does not result in enhanced plasma half-life if this region is not involved in plasma clearance. Indeed, the N-terminal region of the FVIII light chain is already involved in the binding of von Willebrand factor (vWF), a carrier protein critical for the survival of FVIII in circulation. The critical binding to vWF can be disrupted or attenuated by N-terminal modification of Factor VIII. Thus, N-terminal PEGylation of FVIII may have the opposite effect of reducing the plasma half-life of FVIII.
WO90/12874公开了人IL-3、粒细胞集落刺激因子和促红细胞生成素多肽的位点特异性的修饰，这如下实现：插入半胱氨酸，或用半胱氨酸替代另一种氨基酸，然后加入具有巯基反应基团的配体。该配体选择性地偶联半胱氨酸残基。没有公开FVIII或其任何变体的修饰。WO90/12874 discloses site-specific modification of human IL-3, granulocyte colony stimulating factor and erythropoietin polypeptides, which is achieved by inserting a cysteine, or by replacing another amino acid with a cysteine , followed by the addition of ligands with thiol-reactive groups. This ligand selectively couples to cysteine residues. No modification of FVIII or any variant thereof is disclosed.
鉴于上述原因，仍然需要具有更大的体内作用持续时间和降低的免疫原性、同时保持功能活性的改良的FVIII变体。此外，希望以一致的方式将这样的蛋白生产为均质产物。For the above reasons, there remains a need for improved FVIII variants with greater duration of action in vivo and reduced immunogenicity, while maintaining functional activity. Furthermore, it is desirable to produce such proteins in a consistent manner as a homogeneous product.
发明内容Contents of the invention
本发明的一个目的是，提供生物相容的聚合物-缀合的功能性FVIII多肽，其具有提高的药物代谢动力学特性和治疗特性。It is an object of the present invention to provide biocompatible polymer-conjugated functional FVIII polypeptides with improved pharmacokinetic and therapeutic properties.
本发明的另一个目的是，提供生物相容的聚合物-缀合的B结构域缺失的FVIII蛋白，其具有提高的药物代谢动力学特性。Another object of the present invention is to provide biocompatible polymer-conjugated B domain-deleted FVIII proteins with improved pharmacokinetic properties.
本发明的另一个目的是，提供生物相容的聚合物-缀合的功能性FVIII多肽，其具有降低的与低密度脂蛋白受体-相关蛋白(LRP)、低密度脂蛋白(LDL)受体、硫酸乙酰肝素蛋白聚糖(HSPG)和/或针对FVIII的抑制性抗体的结合。Another object of the present invention is to provide biocompatible polymer-conjugated functional FVIII polypeptides with reduced receptor-related protein (LRP), low-density lipoprotein (LDL) receptor Binding of heparan sulfate proteoglycan (HSPG) and/or inhibitory antibodies against FVIII.
本发明的另一个目的是，提供改良的FVIII变体，其具有更大的体内作用持续时间和降低的免疫原性，其能以一致的方式生产为均质产物。Another object of the present invention is to provide improved FVIII variants with greater duration of action in vivo and reduced immunogenicity, which can be produced in a consistent manner as homogeneous products.
在本发明的一个方面，提供了具有因子VIII前凝血剂活性的缀合物，其包含在多肽上的一个或多个预定位点共价附着到一个或多个生物相容的聚合物的功能因子VIII多肽，其中所述预定位点不是N-末端胺。本发明也包括制备该缀合物的方法，该方法包含：突变编码功能因子VIII多肽的核苷酸序列，以在预定位点用半胱氨酸残基的编码序列替代；表达突变的核苷酸序列，以产生半胱氨酸增强的突变蛋白；纯化该突变蛋白；使突变蛋白与生物相容的聚合物反应，所述聚合物已经被激活，以基本上仅在导入的半胱氨酸残基处与多肽反应，从而形成缀合物；和纯化该缀合物。本发明也涉及包含所述缀合物和可药用佐剂的药物组合物和治疗血友病的方法，所述方法通过施用治疗有效量的这些药物组合物给需要的哺乳动物来实现。In one aspect of the invention there is provided a conjugate having Factor VIII procoagulant activity comprising the function of covalently attaching one or more biocompatible polymers at one or more predetermined sites on the polypeptide A Factor VIII polypeptide, wherein the predetermined site is not an N-terminal amine. The present invention also includes a method of preparing the conjugate, the method comprising: mutating the nucleotide sequence encoding a functional Factor VIII polypeptide to replace the coding sequence with a cysteine residue at a predetermined position; expressing the mutated nucleoside acid sequence to produce a cysteine-enhanced mutein; purify the mutein; react the mutein with a biocompatible polymer that has been activated to substantially only react with the introduced cysteine reacting the polypeptide at the residue to form a conjugate; and purifying the conjugate. The invention also relates to pharmaceutical compositions comprising said conjugates and pharmaceutically acceptable adjuvants and methods of treating hemophilia by administering a therapeutically effective amount of these pharmaceutical compositions to a mammal in need thereof.
本发明也涉及因子VIII突变蛋白的位点定向PEG化的方法，该方法包含：(a)表达位点定向因子VIII突变蛋白，其中所述突变蛋白具有在因子VIII突变蛋白的暴露表面上的氨基酸残基的半胱氨酸替代，且该半胱氨酸被加帽；(b)在温和地还原半胱氨酸突变蛋白和释放帽的条件下，使半胱氨酸突变蛋白接触还原剂；(c)从半胱氨酸突变蛋白去除帽和还原剂；和(d)去除还原剂后至少约5分钟，在产生PEG化的因子VIII突变蛋白的条件下，用包含巯基偶联部分的PEG处理半胱氨酸突变蛋白。The present invention also relates to a method of site-directed PEGylation of a Factor VIII mutein, the method comprising: (a) expressing a site-directed Factor VIII mutein, wherein the mutein has an amino acid on an exposed surface of the Factor VIII mutein Cysteine substitution of residues, and the cysteine is capped; (b) exposing the cysteine mutein to a reducing agent under conditions that gently reduce the cysteine mutein and release the cap; (c) removal of the cap and reducing agent from the cysteine mutein; and (d) at least about 5 minutes after removal of the reducing agent, PEGylated factor VIII mutein is treated with PEG comprising a sulfhydryl coupling moiety under conditions that produce a PEGylated Factor VIII mutein Handling of cysteine muteins.
附图说明Description of drawings
图1.PEG突变蛋白的载体图谱和诱变策略。Figure 1. Vector map and mutagenesis strategy for PEG muteins.
图2.经单克隆FVIII抗体层析柱纯化的PEG2蛋白关于时间的280nm UV吸光度谱。使用来自Amersham Bioscience的Explorer 100层析系统，进行层析。Figure 2. 280nm UV absorbance spectrum of PEG2 protein purified by monoclonal FVIII antibody chromatography column with respect to time. Using from Amersham Bioscience Explorer 100 chromatography system, for chromatography.
图3三步位点定向PEG化方法。PEG代表着半胱氨酸-反应性PEG，如PEG-马来酰亚胺。封闭条代表着二硫键形成，而开口条代表着还原的半胱氨酸。Figure 3 Three-step site-directed PEGylation method. PEG stands for cysteine-reactive PEG, such as PEG-maleimide. Closed bars represent disulfide bond formation, while open bars represent reduced cysteine.
图4.PEG2的位点定向PEG化。Figure 4. Site-directed PEGylation of PEG2.
图5.PEG6的位点定向PEG化。Figure 5. Site-directed PEGylation of PEG6.
图6a.BDD、PEG2、4、5和6的位点定向PEG化。上图用重(H)链抗体染色，而下图用轻(L)链抗体染色。“U”是未经加工的含有H和L两者的材料。Figure 6a. Site-directed PEGylation of BDD, PEG2, 4, 5 and 6. The upper panel was stained with the heavy (H) chain antibody, while the lower panel was stained with the light (L) chain antibody. "U" is unprocessed material containing both H and L.
图6b.PEG15和PEG7的PEG化，用PEG2和PEG6作为对照。用TCEP还原起始纯化的PEG突变蛋白(“S”)，并在去除还原剂后，用12kD(“12”)或22kD(“22”)PEG进行PEG化(“R”)。在6％Tris-甘氨酸SDS PAGE上电泳样品，并用重链(“HC”)抗体(左图)或轻链(“LC”)抗体(右图)染色。“U”是未经加工的含有HC和LC两者的材料。用点突出显示PEG化的带。Figure 6b. PEGylation of PEG15 and PEG7, with PEG2 and PEG6 as controls. Initially purified PEG muteins ("S") were reduced with TCEP and, after removal of the reducing agent, PEGylated ("R") with 12kD ("12") or 22kD ("22") PEG. Samples were electrophoresed on 6% Tris-glycine SDS PAGE and stained with heavy chain ("HC") antibody (left panel) or light chain ("LC") antibody (right panel). "U" is unprocessed material containing both HC and LC. The PEGylated bands are highlighted with dots.
图6c.PEG2+6的PEG化，用PEG2和PEG6作为对照。用TCEP还原PEG2、PEG6或PEG2+6，并在去除还原剂后，用5kD(“5”)或43kD(“43”)PEG进行PEG化(“R”)。还用12、22和33kD PEG将PEG2+6 PEG化。在6％Tris-甘氨酸SDS PAGE上电泳样品，并在左图用针对蛋白的考马斯或用重链(H)或轻链(L)抗体进行染色。“U”是未经加工的含有H和L两者的材料。用点突出显示PEG化的带。Figure 6c. PEGylation of PEG2+6 with PEG2 and PEG6 as controls. PEG2, PEG6, or PEG2+6 were reduced with TCEP and, after removal of the reducing agent, PEGylated ("R") with 5kD ("5") or 43kD ("43") PEG. PEG2+6 was also PEGylated with 12, 22 and 33kD PEG. Samples were electrophoresed on 6% Tris-glycine SDS PAGE and stained with Coomassie against the protein or with heavy (H) or light (L) chain antibodies on the left panel. "U" is unprocessed material containing both H and L. The PEGylated bands are highlighted with dots.
图6d.野生型全长FVIII(KG-2)的PEG化，用PEG2作为对照。左凝胶用考马斯染料对蛋白进行染色，而右凝胶用碘对PEG进行染色。“BDD U”是未经加工的含有H和L两者的BDD材料。用点突出显示PEG化的带。Figure 6d. PEGylation of wild-type full-length FVIII (KG-2), using PEG2 as a control. The left gel was stained with Coomassie stain for protein, while the right gel was stained with iodine for PEG. "BDD U" is unprocessed BDD material containing both H and L. The PEGylated bands are highlighted with dots.
图7.PEG化的PEG2的凝血酶切割。A2结构域的N-末端一半颜色为蓝色，而C-末端一半为绿色，R8B12抗体表位突出显示为深绿色(正确的FVIII模型)。用凝血酶处理PEG2(泳道1)和22kD PEG化的PEG2(泳道2)(分别是泳道3和4)，然后在7％Tris-乙酸盐凝胶(Invitrogen)上电泳，并用R8B12抗体染色。每个泳道含有约50ng FVIII。Figure 7. Thrombin cleavage of PEGylated PEG2. The N-terminal half of the A2 domain is colored blue, while the C-terminal half is green, and the R8B12 antibody epitope is highlighted in dark green (correct FVIII model). PEG2 (lane 1) and 22kD PEGylated PEG2 (lane 2) were treated with thrombin (lanes 3 and 4, respectively), followed by electrophoresis on a 7% Tris-acetate gel (Invitrogen) and stained with the R8B12 antibody. Each lane contained approximately 50 ng of FVIII.
图8.PEG化的野生型全长FVIII(KG-2)的凝血酶切割。“S”＝起始KG-2材料。“R”＝还原的KG-2，且去除了还原剂。“P”＝用43kD PEG进行PEG化的“R”。“Pure”＝从过量PEG纯化出来的“P”。“L”＝轻链。用点突出显示PEG化的带。Figure 8. Thrombin cleavage of PEGylated wild-type full-length FVIII (KG-2). "S" = starting KG-2 material. "R" = reduced KG-2 with reducing agent removed. "P" = "R" PEGylated with 43kD PEG. "Pure" = "P" purified from excess PEG. "L" = light chain. The PEGylated bands are highlighted with dots.
图9.PEG化的PEG2的碘染色。在6％Tris甘氨酸凝胶上电泳22或43kD PEG化的PEG2，并用R8B12 FVIII抗体(泳道1和2)或碘(泳道3和4)染色。根据它们的分子量标记泳道，排列2种染色。泳道1和2各自含有约30ng FVIII，而泳道3和4含有约2μg。Figure 9. Iodine staining of PEGylated PEG2. 22 or 43 kD PEGylated PEG2 was run on a 6% Tris glycine gel and stained with R8B12 FVIII antibody (lanes 1 and 2) or iodine (lanes 3 and 4). Rank the 2 stains according to their molecular weight marker lanes. Lanes 1 and 2 each contained approximately 30 ng of FVIII, while lanes 3 and 4 contained approximately 2 μg.
图10.PEG化的和未PEG化的PEG2的MALDI质谱法分析。在PEG2(图10a)或22kD PEG化的PEG2(图10b)上进行MALDI质谱法。PEG化后，PEG2的重(H)链峰显著减少，且出现新峰(H+PEG)，其集中在111kD(22kD PEG+89kD重链)。没有检测到PEG化的轻(L)链峰，预期它集中在100kD(22kD PEG+83kD轻链)。Figure 10. MALDI mass spectrometry analysis of PEGylated and non-PEGylated PEG2. MALDI mass spectrometry was performed on PEG2 (Figure 10a) or 22kD PEGylated PEG2 (Figure 10b). After PEGylation, the heavy (H) chain peak of PEG2 decreased significantly, and a new peak (H+PEG) appeared, which was concentrated at 111 kD (22kD PEG+89kD heavy chain). No PEGylated light (L) chain peak was detected, which was expected to be centered at 100 kD (22 kD PEG + 83 kD light chain).
图11.凝血酶切割后PEG化的和未PEG化的PEG2的MALDI质谱法。Figure 11. MALDI mass spectrometry of PEGylated and non-PEGylated PEG2 after thrombin cleavage.
图12.凝血酶切割之前和之后PEG化的PEG6的MALDI质谱法。Figure 12. MALDI mass spectrometry of PEGylated PEG6 before and after thrombin cleavage.
图13.在尺寸排阻柱上纯化的PEG化的PEG2的280nm UV吸收谱。Figure 13. 280nm UV absorption spectrum of PEGylated PEG2 purified on a size exclusion column.
图14.在阳离子交换柱上纯化的PEG化的和未PEG化的PEG6的280nm UV吸收谱。Figure 14. 280nm UV absorption spectra of PEGylated and non-PEGylated PEG6 purified on a cation exchange column.
图15.在尺寸排阻柱上纯化的PEG化的和未PEG化的PEG6的280nm UV吸收谱。Figure 15. 280nm UV absorption spectra of PEGylated and non-PEGylated PEG6 purified on a size exclusion column.
图16.通过生色测定和凝结测定测得的与未PEG化的蛋白活性相比PEG化的蛋白活性。纯化的全长FVIII表示为KG-2。通过用还原和去除还原剂之后PEG处理的样品的值除以缓冲对照处理的样品的值，其中考虑PEG化得率，确定报告的百分比活性。Figure 16. PEGylated protein activity compared to non-PEGylated protein activity measured by chromogenic assay and coagulation assay. Purified full-length FVIII is denoted KG-2. The reported percent activity was determined by dividing the value of the PEG-treated sample by the value of the buffer control-treated sample after reduction and removal of the reducing agent, taking into account the PEGylation yield.
图17.与PEG2相比，PEG化的PEG2的兔PK研究。Figure 17. Rabbit PK study of PEGylated PEG2 compared to PEG2.
图18.与BDD和PEG2相比，PEG化的PEG2的兔PK研究。在PEG化的PEG2和BDD之间对比P-值。Figure 18. Rabbit PK study of PEGylated PEG2 compared to BDD and PEG2. P-values were compared between PEGylated PEG2 and BDD.
图19.与BDD和PEG6相比，PEG化的PEG6的兔PK研究。Figure 19. Rabbit PK study of PEGylated PEG6 compared to BDD and PEG6.
图20.与未修饰的fl FVIII相比，PEG化的野生型全长(“fl”)FVIII的兔PK研究。Figure 20. Rabbit PK study of PEGylated wild-type full-length ("fl") FVIII compared to unmodified fl FVIII.
图21.与PEG6和BDD相比，PEG化的PEG6的血友病小鼠PK研究。Figure 21. Hemophilia mouse PK study of PEGylated PEG6 compared to PEG6 and BDD.
图22.与BDD相比，22和43kD PEG化的PEG2的正常小鼠PK研究。Figure 22. Normal mouse PK study of 22 and 43 kD PEGylated PEG2 compared to BDD.
图23.与BDD相比，22kD PEG化的PEG2的全时程正常小鼠PK研究。Figure 23. Full time normal mouse PK study of 22kD PEGylated PEG2 compared to BDD.
图24.血友病小鼠(BDD)因子VIII回收柱状图，它描绘了在血友病小鼠测定中两种BDD因子VIII的半衰期的药物代谢动力学(PK)评价。Figure 24. Hemophilia mouse (BDD) Factor VIII recovery histogram depicting the pharmacokinetic (PK) evaluation of the half-life of two BDD Factor VIII in the hemophilia mouse assay.
图25.与BDD相比，22kD PEG化的PEG2的血友病小鼠肾撕裂伤研究。载体处理的小鼠具有25uL/g体重的失血。Figure 25. Hemophilia mouse kidney tear study of 22kD PEGylated PEG2 compared to BDD. Vehicle-treated mice had a blood loss of 25 uL/g body weight.
图26.在有递增量的FVIII抗体存在下，PEG化的PEG2和BDD的生色活性。抗体表位在括号中指明。Figure 26. Chromogenic activity of PEGylated PEG2 and BDD in the presence of increasing amounts of FVIII antibody. Antibody epitopes are indicated in parentheses.
图27.在有递增量的FVIII mAB 413抗体存在下，PEG化的PEG2的生色活性。Figure 27. Chromogenic activity of PEGylated PEG2 in the presence of increasing amounts of FVIII mAB 413 antibody.
图28.在有源自已经发展对FVIII的抑制剂的患者的人血浆存在下，BDD、43kD PEG化的PEG2、33kD PEG化的PEG6和33kD双PEG化(diPEGylation)的PEG2+6的生色活性。在上面记录了抑制剂效价和血液收集日期。上面的2个图包括从5-405倍患者血浆稀度收集的数据。左下图集中在患者HRF-828血浆的1∶15倍稀度。右下图证实，为上面的2个图中的每个FVIII样品使用的0.064IU/mL不是饱和剂量。Figure 28. Chromogenicity of BDD, 43kD PEGylated PEG2, 33kD PEGylated PEG6 and 33kD diPEGylation PEG2+6 in the presence of human plasma derived from patients who have developed inhibitors to FVIII active. Inhibitor titers and blood collection dates are recorded above. The upper 2 graphs include data collected from 5-405 times patient plasma dilutions. The lower left panel focuses on the 1:15 dilution of patient HRF-828 plasma. The lower right graph demonstrates that the 0.064 IU/mL used for each FVIII sample in the upper 2 graphs was not a saturating dose.
图29.PEG化筛选方法和验证。上图显示了瞬时表达的PEG突变蛋白的PEG化筛选示意图。下图显示了使用重链(“H”)-特异性的抗体(左)或轻链(“L”)特异性的抗体(右)对PEG化的产物的蛋白印迹分析。用点突出显示PEG化的带。“U”是未经加工的含有H和L两者的材料。Figure 29. PEGylation screening method and validation. The upper panel shows a schematic diagram of PEGylation screening of transiently expressed PEG muteins. The lower panel shows Western blot analysis of PEGylated products using heavy chain ("H")-specific antibodies (left) or light chain ("L") specific antibodies (right). The PEGylated bands are highlighted with dots. "U" is unprocessed material containing both H and L.
图30.PEG15-17的PEG化筛选。使用重链(“H”)-特异性的抗体(R8B12和58.12)或轻链(“L”)特异性的抗体(C7F7和GM)，对PEG化的产物进行蛋白印迹分析。所有3种突变蛋白都是对重链选择性的，相对PEG化效率是PEG15～PEG16＞PEG17。用点突出显示PEG化的带。“U”是未经加工的含有H和L两者的材料。Figure 30. PEGylation screen for PEG15-17. Western blot analysis of PEGylated products was performed using heavy chain ("H")-specific antibodies (R8B12 and 58.12) or light chain ("L") specific antibodies (C7F7 and GM). All three muteins are selective for the heavy chain with a relative PEGylation efficiency of PEG15-PEG16 > PEG17. The PEGylated bands are highlighted with dots. "U" is unprocessed material containing both H and L.
图31.该凝胶显示了作为还原剂浓度函数的PEG2+14的PEG化。在4℃，用67-670uM的TCEP处理PEG2+14 30分钟。通过旋转柱去除还原剂，随后用12kD PEG进行PEG化。FVIII的重链和轻链分别突出显示为“H”和“L”。2个印迹指出了PEG化的重链和轻链。Figure 31. This gel shows PEGylation of PEG2+14 as a function of reducing agent concentration. PEG2+14 was treated with TCEP at 67-670uM for 30 minutes at 4°C. The reducing agent was removed by spin column followed by PEGylation with 12kD PEG. The heavy and light chains of FVIII are highlighted as "H" and "L", respectively. 2 blots indicate PEGylated heavy and light chains.
图32.用67-670uM TCEP处理、随后去除还原剂的PEG2+14的解卷积质谱。Figure 32. Deconvoluted mass spectrum of PEG2+14 treated with 67-670uM TCEP followed by reducing agent removal.
具体实施方式detailed description
本发明是基于下述发现，即具有FVIII活性的多肽可以在非N-末端胺的预定位点共价附着到生物相容的聚合物上，且这样的多肽基本上保留它们的凝血剂活性。此外，这些多肽缀合物具有提高的循环时间和降低的抗原性。本发明的缀合物胜过现有技术的具有随机附着到FVIII或附着在N-末端的聚合物的缀合物。位点定向附着允许人们设计避过生物学活性所需区域并从而维持相当大的FVIII活性的修饰。它也允许设计成附着聚合物，来阻断在参与FVIII清除的位点的结合。位点定向结合也允许通过随机的聚合物偶联，生成均匀的产物，而不是现有技术生成的异质缀合物。通过避免在轻链N-末端胺处的附着，本发明的缀合物避免了因为将配体附着在FVIII多肽的活性位点可能产生的活性损失。认为轻链N-末端区域参与vWF因子与FVIII的结合，这是循环中的稳定结合。The present invention is based on the discovery that polypeptides having FVIII activity can be covalently attached to biocompatible polymers at predetermined sites other than the N-terminal amine, and that such polypeptides substantially retain their coagulant activity. Furthermore, these Polypeptide Conjugates have improved circulation time and reduced antigenicity. The conjugates of the present invention outperform prior art conjugates with polymers randomly attached to FVIII or attached to the N-terminus. Site-directed attachment allows one to design modifications that avoid regions required for biological activity and thereby maintain substantial FVIII activity. It also allows the design of attachment polymers that block binding at sites involved in FVIII clearance. Site-directed conjugation also allows for random polymer coupling, resulting in homogeneous products rather than the heterogeneous conjugates produced by prior art techniques. By avoiding attachment at the N-terminal amine of the light chain, the conjugates of the present invention avoid the loss of activity that may occur by attaching the ligand to the active site of the FVIII polypeptide. The N-terminal region of the light chain is thought to be involved in the binding of vWF factor to FVIII, which is a stable binding in circulation.
定义definition
生物相容的聚合物。生物相容的聚合物包括聚环氧烷，例如但不限于聚乙二醇(PEG)、葡聚糖、多聚乙酰神经氨酸或其它基于碳水化合物的聚合物、氨基酸聚合物、生物素衍生物、聚乙烯醇(PVA)、聚羧酸酯、聚乙烯吡咯烷酮、乙烯-马来酸酐共聚物、苯乙烯-苹果酸酐共聚物、聚唑啉、聚丙烯酰吗啉、肝素、清蛋白、纤维素、壳聚糖水解产物、淀粉如羟乙基-淀粉和羟丙基-淀粉、糖原、琼脂糖和其衍生物、瓜耳胶、支链淀粉、菊粉、黄原胶、角叉藻聚糖、果胶、藻酸水解产物、其它生物聚合物和其任意等同物。优选的是聚乙二醇，且更优选的是甲氧基聚乙二醇(mPEG)。其它有用的聚(亚烷基)二醇化合物是聚丙二醇(PPG)、聚丁二醇(PBG)、PEG-缩水甘油醚(Epox-PEG)、PEG-氧羰基咪唑(CDI-PEG)、分支的聚乙二醇、线性的聚乙二醇、分叉的聚乙二醇和多臂的或“超分支的”聚乙二醇(星形-PEG)。biocompatible polymer. Biocompatible polymers include polyalkylene oxides such as, but not limited to, polyethylene glycol (PEG), dextran, polyacetylneuraminic acid or other carbohydrate-based polymers, amino acid polymers, biotin-derived Polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone, ethylene-maleic anhydride copolymer, styrene-malic anhydride copolymer, poly Azoline, polyacrylmorpholine, heparin, albumin, cellulose, chitosan hydrolysates, starches such as hydroxyethyl-starch and hydroxypropyl-starch, glycogen, agarose and its derivatives, guar gum , pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acid hydrolyzate, other biopolymers and any equivalents thereof. Preferred is polyethylene glycol, and more preferred is methoxypolyethylene glycol (mPEG). Other useful poly(alkylene) glycol compounds are polypropylene glycol (PPG), polybutylene glycol (PBG), PEG-glycidyl ether (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), branched polyethylene glycol, linear polyethylene glycol, branched polyethylene glycol, and multi-armed or "hyperbranched" polyethylene glycol (star-PEG).
聚乙二醇(PEG)。“PEG”和“聚乙二醇”在本文中可互换地使用，且包括任意的水溶性的聚环氧乙烷。一般地，根据本发明使用的PEG包含下面的结构“--(OCH2CH2)n--”，其中(n)是2-4000。如本文所使用的，根据末端氧是否已经被置换，PEG也包括“--CH2CH2--O(CH2CH2O)n--CH2CH2--”和“--(OCH2CH2)nO--”。在说明书和权利要求全文中，应当牢记，术语“PEG”包括具有各种末端或“封端”基团的结构，例如但不限于羟基或C1-20烷氧基。术语“PEG”也指含有大多数(即大于50％)的-OCH2CH2-重复亚基的聚合物。关于具体形式，PEG可以具有任意数目的许多分子量，以及结构或几何形状，如分支的、线性的、分叉的、和多功能的。polyethylene glycol (PEG). "PEG" and "polyethylene glycol" are used interchangeably herein and include any water-soluble polyethylene oxide. Generally, the PEG used according to the present invention comprises the following structure "--(OCH2CH2) n--", wherein (n ) is 2-4000. As used herein, PEG also includes "--CH2CH2-- O(CH2CH2O)n--CH2CH2-- " and "--(OCH2 CH2 )n O--". Throughout the specification and claims, it should be kept in mind that the term "PEG" includes structures with various terminal or "capping" groups such as, but not limited to, hydroxyl or C1-20 alkoxy. The term "PEG" also refers to polymers that contain a majority (ie, greaterthan 50%) of repeating subunits of -OCH2CH2-. With regard to specific forms, PEG can have any number of many molecular weights, as well as structures or geometries, such as branched, linear, forked, and multifunctional.
PEG化。PEG化是聚乙二醇(PEG)共价附着到诸如蛋白的分子上的过程。PEGylation. PEGylation is the process by which polyethylene glycol (PEG) is covalently attached to molecules such as proteins.
激活的或有活性的官能团。当将诸如生物相容的聚合物的官能团描述为激活的时，该官能团可以容易地与其它分子上的亲电子试剂或亲核试剂反应。Activated or reactive functional groups. When a functional group such as a biocompatible polymer is described as activated, the functional group can readily react with electrophiles or nucleophiles on other molecules.
B结构域缺失的FVIII(BDD)。如本文所使用的，BDD的特征在于，具有含有FVIII的B-结构域的14个氨基酸以外的所有缺失的氨基酸序列。B-结构域的前4个氨基酸(SFSQ，SEQID NO：1)连接到B-结构域的最后10个残基上(NPPVLKRHQR，SEQ ID NO：2)(Lind，P.等，1995，Eur.J.Biochem.232，pp.19-27)。本文使用的BDD具有SEQ ID NO：3的氨基酸序列。B-domain deleted FVIII (BDD). As used herein, a BDD is characterized by having an amino acid sequence that contains deletions of all but 14 amino acids of the B-domain of FVIII. The first 4 amino acids of the B-domain (SFSQ, SEQ ID NO: 1) are linked to the last 10 residues of the B-domain (NPPVLKRHQR, SEQ ID NO: 2) (Lind, P. et al., 1995, Eur. J. Biochem. 232, pp. 19-27). BDD as used herein has the amino acid sequence of SEQ ID NO:3.
FVIII.凝血因子VIII(FVIII)是在由肝合成并释放进血流中的糖蛋白。在循环血液中，它结合von Willebrand因子(vWF，也称作因子VIII-相关抗原)，以形成稳定的复合物。被凝血酶激活后，它从复合物解离，以与凝结级联中的其它凝血因子相互作用，这最终导致血栓的形成。人全长FVIII具有SEQ ID NO：4的氨基酸序列，尽管等位基因变体是可能的。FVIII. Factor VIII (FVIII) is a glycoprotein that is synthesized by the liver and released into the bloodstream. In circulating blood, it binds von Willebrand factor (vWF, also known as Factor VIII-related antigen) to form a stable complex. Upon activation by thrombin, it dissociates from the complex to interact with other coagulation factors in the coagulation cascade, which ultimately leads to thrombus formation. Human full-length FVIII has the amino acid sequence of SEQ ID NO: 4, although allelic variants are possible.
功能因子VIII多肽。如本文所使用的，功能因子VIII多肽表示功能多肽或多肽的组合，其能体内或体外地改正人因子VIII缺陷，所述缺陷的特征在于例如血友病A。因子VIII具有许多种自然状态的降解或加工形式。它们蛋白水解地源自前体，即一种链蛋白，如本文所证实的。功能因子VIII多肽包括这样的单链蛋白，且还提供了这些具有改正人因子VIII缺陷的生物学活性的各种降解产物。可能存在等位基因变体。功能因子VIII多肽包括所有这样的等位基因变体、糖基化的形式、导致因子VIII的衍生物的修饰和片段，只要它们含有人因子VIII的功能片段，且同样基本的特征性人因子VIII功能活性保持未受影响。通过本文所述的简单的体外测试，可以容易地鉴别出具有必需的功能活性的那些因子VIII衍生物。此外，功能因子VIII多肽能在因子IXa、钙和磷脂存在下，催化因子X向Xa的转化，以及改正源自患血友病A个体的血浆中的凝结缺陷。从本文的人因子VIII氨基酸序列和功能区域的序列公开内容，本领域技术人员会明白可以通过DNA的限制酶切割或人因子VIII蛋白的蛋白水解或其它降解衍生的片段。Functional Factor VIII polypeptides. As used herein, a functional Factor VIII polypeptide refers to a functional polypeptide or combination of polypeptides that is capable of correcting human Factor VIII deficiency, characteristic of, for example, hemophilia A, in vivo or in vitro. Factor VIII has many degraded or processed forms in its natural state. They are proteolytically derived from a precursor, a catenin, as demonstrated herein. Functional Factor VIII polypeptides include such single-chain proteins, and various degradation products of these having biological activity to correct human Factor VIII deficiency are also provided. Allelic variants may exist. Functional Factor VIII polypeptides include all such allelic variants, glycosylated forms, modifications and fragments leading to derivatives of Factor VIII, as long as they contain functional fragments of human Factor VIII, and also essentially the characteristic human Factor VIII Functional activity remained unaffected. Those Factor VIII derivatives possessing the requisite functional activity can readily be identified by simple in vitro assays as described herein. In addition, functional Factor VIII polypeptides can catalyze the conversion of Factor X to Xa in the presence of Factor IXa, calcium, and phospholipids, as well as correct coagulation defects in plasma derived from individuals with hemophilia A. From the amino acid sequence of human factor VIII and the sequence disclosure of functional regions herein, those skilled in the art will understand that fragments can be derived by restriction enzyme cleavage of DNA or proteolysis or other degradation of human factor VIII protein.
FIX。如本文所使用的，FIX指凝结因子IX，它也称作人凝血因子IX，或血浆促凝血酶原激酶组分。FIX. As used herein, FIX refers to coagulation factor IX, which is also known as human coagulation factor IX, or the plasma thromboplastin component.
FX。如本文所使用的，FX指凝结因子X，它也称作人凝血因子X和Stuart-Prower因子。FX. As used herein, FX refers to coagulation factor X, which is also known as human coagulation factor X and Stuart-Prower factor.
药物代谢动力学。“药物代谢动力学”(“PK”)是用于描述药物在身体中的吸收、分布、代谢和消除的性质的术语。药物的药物代谢动力学的提高，指使药物在体内更有效地作为治疗剂的这些特征(尤其是它在身体中的有效持续时间)的提高。Pharmacokinetic. "Pharmacokinetics" ("PK") is a term used to describe the properties of the absorption, distribution, metabolism and elimination of drugs in the body. Improvements in the pharmacokinetics of a drug refer to improvements in those characteristics of a drug that make it more effective in the body as a therapeutic agent, especially its duration of effectiveness in the body.
突变蛋白。突变蛋白是一种基因工程改造的蛋白，其源自实验室诱导的蛋白或多肽的突变。mutant protein. A mutein is a genetically engineered protein that results from laboratory-induced mutations in a protein or polypeptide.
蛋白。如本文所使用的，蛋白和多肽是同义词。protein. As used herein, protein and polypeptide are synonymous.
FVIII清除受体。本文所使用的FVIII清除受体指功能性FVIII多肽上的受体区域，其结合或联合一个或多个其它分子，以导致从循环清除FVIII。因子VIII清除受体包括但不限于FVIII分子的结合LRP、LDL受体和/或HSPG的区域。FVIII clears receptors. As used herein, a FVIII clearance receptor refers to a receptor region on a functional FVIII polypeptide that binds or associates with one or more other molecules to result in clearance of FVIII from circulation. Factor VIII scavenging receptors include, but are not limited to, regions of the FVIII molecule that bind LRP, LDL receptors, and/or HSPG.
讨论discuss
根据本发明的方法预见到，可以在预定位点突变任何功能因子VIII多肽，然后在该位点共价附着生物相容的聚合物。有用的多肽包括但不限于，具有SEQ ID NO：4所示的氨基酸序列的全长因子VIII，和具有SEQ ID NO：3所示的氨基酸序列的BDD FVIII。优选的是BDD FVIII。The method according to the invention envisions that any functional Factor VIII polypeptide can be mutated at a predetermined site and then covalently attached to a biocompatible polymer at that site. Useful polypeptides include, but are not limited to, full-length Factor VIII having the amino acid sequence set forth in SEQ ID NO:4, and BDD FVIII having the amino acid sequence set forth in SEQ ID NO:3. Preferred is BDD FVIII.
在本发明的缀合物中使用的生物相容的聚合物可以是上面讨论的任意聚合物。选择生物相容的聚合物，以提供所需的药物代谢动力学的提高。例如，选择聚合物的特性、大小和结构，从而提高具有FVIII活性的多肽的循环半衰期，或降低多肽的抗原性，而没有不可接受的活性降低。优选地，聚合物包含PEG，且更优选地其至少50％的分子量是PEG。在一个实施方案中，聚合物是在末端用封端部分(如羟基、烷氧基、取代的烷氧基、链烯氧基、取代的链烯氧基、炔氧基(alkynoxy)、取代的炔氧基、芳基氧和取代的芳基氧)封端的聚乙二醇。更优选的是包含甲氧基聚乙二醇的聚合物。更优选的是包含甲氧基聚乙二醇的聚合物，其大小范围为3kD-100kD，且更优选地5kD-64kD或5kD-43kD。The biocompatible polymer used in the conjugates of the invention can be any of the polymers discussed above. Biocompatible polymers are selected to provide the desired pharmacokinetic enhancement. For example, the properties, size and structure of the polymer are selected to increase the circulating half-life of a polypeptide having FVIII activity, or to reduce the antigenicity of the polypeptide without unacceptable reduction in activity. Preferably, the polymer comprises PEG, and more preferably at least 50% of its molecular weight is PEG. In one embodiment, the polymer is terminally substituted with a capping moiety such as hydroxyl, alkoxy, substituted alkoxy, alkenyloxy, substituted alkenyloxy, alkynoxy, Alkynyloxy, aryloxy and substituted aryloxy) terminated polyethylene glycols. More preferred are polymers comprising methoxypolyethylene glycol. More preferred are polymers comprising methoxypolyethylene glycol in the size range of 3kD-100kD, and more preferably 5kD-64kD or 5kD-43kD.
优选地，聚合物具有活性部分。例如，在一个实施方案中，聚合物具有巯基活性部分，其可以与功能因子VIII多肽上的游离半胱氨酸反应，以形成共价键。这样的巯基活性部分包括硫羟、三氟甲磺酸(triflate)、2，2，2-三氟乙磺酸(tresylate)、氮丙啶、环氧乙烷、S-吡啶基或马来酰亚胺部分。优选的是马来酰亚胺部分。在一个实施方案中，聚合物是线性的，且在与巯基不强烈反应的一个末端具有“帽”(如甲氧基)，而在另一个末端具有巯基活性部分。在一个优选的实施方案中，缀合物包含PEG-马来酰亚胺，且大小范围为5kD-64kD。Preferably, the polymer has a reactive moiety. For example, in one embodiment, the polymer has a sulfhydryl reactive moiety that can react with a free cysteine on a functional Factor VIII polypeptide to form a covalent bond. Such thiol-reactive moieties include thiol, triflate, 2,2,2-trifluoroethanesulfonate (tresylate), aziridine, oxirane, S-pyridyl or maleyl imine moiety. Preferred are maleimide moieties. In one embodiment, the polymer is linear and has a "cap" (eg, methoxy) at one end that is not strongly reactive with thiol groups, and a thiol-reactive moiety at the other end. In a preferred embodiment, the conjugate comprises PEG-maleimide and has a size ranging from 5kD to 64kD.
在下面的实施例中，提供了选择有用的生物相容的聚合物的其它指导。In the Examples below, additional guidance for selecting useful biocompatible polymers is provided.
通过本领域已知的任意方法，可以发生编码具有FVIII活性的多肽的核苷酸序列的位点定向突变。优选的方法包括在选择的用于共价附着聚合物的位点处导入半胱氨酸密码子的诱变。这可以如下实现：使用可商业得到的位点定向诱变试剂盒，如StratagenecQuickChangeTM II位点定向诱变试剂盒、Clontech Transformer位点定向诱变试剂盒号K1600-1、Invitrogen GenTaylor位点定向诱变系统号12397014、Promega Altered SitesII体外诱变系统试剂盒号Q6210或Takara Mirus Bio LA PCR诱变试剂盒号TAK RR016。Site-directed mutagenesis of a nucleotide sequence encoding a polypeptide having FVIII activity can occur by any method known in the art. A preferred method involves mutagenesis to introduce cysteine codons at selected sites for covalent attachment of the polymer. This can be achieved by using commercially available site-directed mutagenesis kits such as StratagenecQuickChange™ II Site-Directed Mutagenesis Kit, Clontech Transformer Site-Directed Mutagenesis Kit No. K1600-1, Invitrogen GenTaylor Site-Directed Mutagenesis Mutation system number 12397014, Promega Altered Sites II in vitro mutagenesis system kit number Q6210 or Takara Mirus Bio LA PCR mutagenesis kit number TAK RR016.
本发明的缀合物可以如下制备：首先用半胱氨酸密码子替代功能性FVIII多肽表面上的一个或多个氨基酸的密码子，在重组表达系统中生产半胱氨酸突变蛋白，使突变蛋白与半胱氨酸-特异性的聚合物试剂反应，和纯化突变蛋白。The conjugate of the present invention can be prepared as follows: firstly replace the codon of one or more amino acids on the surface of functional FVIII polypeptide with cysteine codon, produce cysteine mutein in recombinant expression system, make mutation The protein is reacted with a cysteine-specific polymer reagent, and the mutein is purified.
在该系统中，在半胱氨酸位点添加聚合物，可以通过聚合物上的马来酰亚胺活性的官能度来实现。在下文提供了该技术的实例。使用的巯基活性聚合物的量应当至少与要衍生的半胱氨酸的摩尔量等摩尔，且优选地过量存在。优选地，使用至少5倍摩尔过量的巯基反应性聚合物，且更优选地，使用至少10倍过量的这种聚合物。用于共价附着的其它条件属于本领域技术人员的技术。In this system, addition of the polymer at the cysteine site can be achieved through maleimide-reactive functionality on the polymer. Examples of this technique are provided below. The amount of thiol-reactive polymer used should be at least equimolar to the molar amount of cysteine to be derivatized, and is preferably present in excess. Preferably, at least a 5-fold molar excess of the thiol-reactive polymer is used, and more preferably, at least a 10-fold excess of such polymer is used. Other conditions for covalent attachment are within the skill of those skilled in the art.
在下面的实施例中，以本领域常规方式命名突变蛋白。命名突变体的惯例是基于SEQ ID NO：4提供的成熟的全长因子VIII的氨基酸序列的。作为一种分泌蛋白，FVIII含有在翻译过程中被蛋白水解地切割的信号序列。去除19氨基酸信号序列后，分泌的FVIII产物的第一个氨基酸是丙氨酸。In the following examples, muteins are named in a manner conventional in the art. The convention for naming mutants is based on the amino acid sequence of mature full-length Factor VIII provided in SEQ ID NO:4. As a secreted protein, FVIII contains a signal sequence that is proteolytically cleaved during translation. After removal of the 19 amino acid signal sequence, the first amino acid of the secreted FVIII product is alanine.
正如常规的和本文使用的，当提及BDD FVIII中的突变氨基酸时，通过其在全长FVIII序列中的位置命名突变氨基酸。例如，下面讨论的PEG6突变蛋白命名为K1808C，因为它将在类似于全长序列的1808位置处的赖氨酸(K)改变成半胱氨酸(C)。As is conventional and used herein, when referring to a mutated amino acid in BDD FVIII, the mutated amino acid is named by its position in the full-length FVIII sequence. For example, the PEG6 mutein discussed below was named K1808C because it changed a lysine (K) to a cysteine (C) at position 1808 similar to the full-length sequence.
用于共价结合聚合物的预定位点最好地选自暴露于多肽表面、不参与FVIII活性或不参与体内稳定化FVIII的其它机理(如结合vWF)的位点。这样的位点还最好地选自已知参与灭活FVIII或从循环中清除FVIII的机理的那些位点。下面详细讨论了这些位点的选择。优选位点包括在下述对象的结合位点处或附近的氨基酸残基：(a)低密度脂蛋白受体相关蛋白，(b)硫酸肝素蛋白聚糖，(c)低密度脂蛋白受体和/或(d)因子VIII抑制性抗体。“在结合位点处或附近”指残基与结合位点足够接近，从而使得生物相容的聚合物与该位点的共价附着导致结合位点的位阻。例如，预期这样的位点是在结合位点的内。Predetermined sites for covalently attaching polymers are preferably selected from sites exposed on the surface of the polypeptide, not involved in FVIII activity, or not involved in other mechanisms of stabilizing FVIII in vivo, such as binding to vWF. Such sites are also preferably selected from those sites known to be involved in mechanisms of inactivating FVIII or clearing FVIII from circulation. The selection of these sites is discussed in detail below. Preferred sites include amino acid residues at or near the binding site for (a) low-density lipoprotein receptor-associated protein, (b) heparan sulfate proteoglycan, (c) low-density lipoprotein receptor and /or (d) Factor VIII inhibitory antibodies. "At or near the binding site" means that the residue is sufficiently close to the binding site such that covalent attachment of a biocompatible polymer to the site results in steric hindrance of the binding site. For example, such a site is expected to be at the binding site Inside.
在本发明的一个实施方案中，生物相容的聚合物共价附着在功能因子VIII多肽的下述位置处或附近的氨基酸残基上：(a)上面定义的因子VIII清除受体，(b)能降解因子VIII的蛋白酶的结合位点，和/或(c)因子VIII抑制性抗体的结合位点。蛋白酶可以为激活的C蛋白(APC)。在另一个实施方案中，生物相容的聚合物共价附着在功能因子VIII多肽的预定位点，从而使得低密度脂蛋白受体相关蛋白与多肽的结合小于与未缀合时的多肽的结合，且优选地，不到1/2。在一个实施方案中，生物相容的聚合物共价附着在功能因子VIII多肽的预定位点，从而使得硫酸肝素蛋白聚糖与多肽的结合小于与未缀合时的多肽的结合，且优选地，不到1/2。在另一个实施方案中，生物相容的聚合物共价附着在功能因子VIII多肽的预定位点，从而使得因子VIII抑制性抗体与多肽的结合小于与未缀合时的多肽的结合，优选地，不到与未缀合时的多肽的结合的1/2。在另一个实施方案中，生物相容的聚合物共价附着在功能因子VIII多肽的预定位点，从而使得低密度脂蛋白受体与多肽的结合小于与未缀合时的多肽的结合，优选地，不到1/2。在另一个实施方案中，生物相容的聚合物共价附着在功能因子VIII多肽的预定位点，从而使得血浆蛋白酶对多肽的降解小于对未缀合时的多肽的降低。在另一个实施方案中，血浆蛋白酶对多肽的降解不到当多肽未缀合时在相同条件下经相同时间段测得的对多肽的降解的1/2。In one embodiment of the invention, a biocompatible polymer is covalently attached to an amino acid residue at or near a functional Factor VIII polypeptide at or near: (a) a Factor VIII scavenging receptor as defined above, (b ) a binding site for a protease capable of degrading Factor VIII, and/or (c) a binding site for a Factor VIII inhibitory antibody. The protease can be activated protein C (APC). In another embodiment, a biocompatible polymer is covalently attached to a predetermined site of a functional Factor VIII polypeptide such that the low density lipoprotein receptor-associated protein binds to the polypeptide less than it binds to the polypeptide when unconjugated , and preferably, less than 1/2. In one embodiment, a biocompatible polymer is covalently attached to a predetermined site of a functional Factor VIII polypeptide such that the binding of the heparan sulfate proteoglycan to the polypeptide is less than that of the polypeptide when unconjugated, and preferably , less than 1/2. In another embodiment, a biocompatible polymer is covalently attached to a predetermined site of a functional Factor VIII polypeptide such that the Factor VIII inhibitory antibody binds to the polypeptide less than it binds to the polypeptide when unconjugated, preferably , less than 1/2 of the binding to the unconjugated polypeptide. In another embodiment, a biocompatible polymer is covalently attached to a predetermined site of a functional Factor VIII polypeptide such that the binding of the low density lipoprotein receptor to the polypeptide is less than that of the unconjugated polypeptide, preferably ground, less than 1/2. In another embodiment, a biocompatible polymer is covalently attached to a predetermined site of a functional Factor VIII polypeptide such that degradation of the polypeptide by plasma proteases is less than that of the unconjugated polypeptide. In another embodiment, plasma proteases degrade the polypeptide less than 1/2 the degradation of the polypeptide measured under the same conditions over the same period of time when the polypeptide is unconjugated.
使用表面等离子体共振技术(Biacore)，可以确定LRP、LDL受体或HSPG对FVIII的结合亲和力。例如，可以将FVIII直接地或通过FVIII抗体间接地涂布在BiacoreTM芯片上，并可以将各种浓度的LRP传递到芯片上，来测量相互作用的启动速率和关闭速率(BovenschenN.等，2003，J.Biol.Chem.278(11)，pp.9370-7)。两个速率的比给出亲和力的度量。需要PEG化后亲和力的2倍，优选地5倍，更优选地10倍，和更优选地30倍的降低。Using surface plasmon resonance technology (Biacore), the binding affinity of LRP, LDL receptor or HSPG to FVIII can be determined. For example, FVIII can be coated on Biacore™ chips directly or indirectly through FVIII antibodies, and various concentrations of LRP can be delivered to the chip to measure the on- and off-rates of the interaction (Bovenschen N. et al., 2003 , J. Biol. Chem. 278(11), pp.9370-7). The ratio of the two rates gives a measure of affinity. A 2-fold, preferably 5-fold, more preferably 10-fold, and more preferably 30-fold reduction in affinity after PEGylation is desired.
通过本领域技术人员已知的任意方法，可以测量蛋白酶APC对FVIII的降解。Degradation of FVIII by the protease APC can be measured by any method known to those skilled in the art.
在一个实施方案中，生物相容的聚合物共价附着在多肽的一个或多个因子VIII氨基酸位置81、129、377、378、468、487、491、504、556、570、711、1648、1795、1796、1803、1804、1808、1810、1864、1903、1911、2091、2118和2284。在另一个实施方案中，生物相容的聚合物共价附着在多肽的一个或多个因子VIII氨基酸位置377、378、468、491、504、556、1795、1796、1803、1804、1808、1810、1864、1903、1911和2284，且(1)缀合物与低密度脂蛋白受体相关蛋白的结合小于未缀合的多肽与低密度脂蛋白受体相关蛋白的结合；(2)缀合物与低密度脂蛋白受体的结合小于未缀合的多肽与低密度脂蛋白受体的结合；或(3)缀合物与低密度脂蛋白受体相关蛋白和低密度脂蛋白受体的结合小于未缀合的多肽与低密度脂蛋白受体相关蛋白和低密度脂蛋白受体的结合。In one embodiment, the biocompatible polymer is covalently attached to one or more of the Factor VIII amino acid positions 81, 129, 377, 378, 468, 487, 491, 504, 556, 570, 711, 1648, 1795, 1796, 1803, 1804, 1808, 1810, 1864, 1903, 1911, 2091, 2118, and 2284. In another embodiment, the biocompatible polymer is covalently attached to one or more of the Factor VIII amino acid positions 377, 378, 468, 491, 504, 556, 1795, 1796, 1803, 1804, 1808, 1810 of the polypeptide , 1864, 1903, 1911 and 2284, and (1) the binding of the conjugate to the low-density lipoprotein receptor-associated protein is less than the binding of the unconjugated polypeptide to the low-density lipoprotein receptor-associated protein; (2) the conjugate The binding of the compound to the low-density lipoprotein receptor is less than the binding of the unconjugated polypeptide to the low-density lipoprotein receptor; or (3) the binding of the conjugate to the low-density lipoprotein receptor-associated protein and the low-density lipoprotein receptor Binding was less than that of unconjugated polypeptide to low density lipoprotein receptor associated protein and low density lipoprotein receptor.
在另一个实施方案中，生物相容的聚合物共价附着在多肽的一个或多个因子VIII氨基酸位置377、378、468、491、504、556和711，且该缀合物与硫酸肝素蛋白聚糖的结合小于未缀合的多肽与硫酸肝素蛋白聚糖的结合。在另一个实施方案中，生物相容的聚合物共价附着在多肽的一个或多个因子VIII氨基酸位置81、129、377、378、468、487、491、504、556、570、711、1648、1795、1796、1803、1804、1808、1810、1864、1903、1911、2091、2118和2284，且该缀合物具有比未缀合的多肽更小的与因子VIII抑制性抗体的结合。在另一个实施方案中，生物相容的聚合物共价附着在多肽的一个或多个因子VIII氨基酸位置81、129、377、378、468、487、491、504、556、570、711、1648、1795、1796、1803、1804、1808、1810、1864、1903、1911、2091、2118和2284，且优选地在一个或多个位置377、378、468、491、504、556和711，且该缀合物具有比未缀合的多肽更少的来自能降解因子VIII的血浆蛋白酶的降解。更优选的，血浆蛋白酶是激活的C蛋白。In another embodiment, a biocompatible polymer is covalently attached to one or more Factor VIII amino acid positions 377, 378, 468, 491, 504, 556, and 711 of the polypeptide, and the conjugate is combined with the heparan sulfate protein The binding of glycans is less than the binding of unconjugated polypeptide to heparan sulfate proteoglycans. In another embodiment, the biocompatible polymer is covalently attached to one or more Factor VIII amino acid positions 81, 129, 377, 378, 468, 487, 491, 504, 556, 570, 711, 1648 of the polypeptide , 1795, 1796, 1803, 1804, 1808, 1810, 1864, 1903, 1911, 2091, 2118, and 2284, and the conjugates had less binding to the Factor VIII inhibitory antibody than the unconjugated polypeptide. In another embodiment, the biocompatible polymer is covalently attached to one or more Factor VIII amino acid positions 81, 129, 377, 378, 468, 487, 491, 504, 556, 570, 711, 1648 of the polypeptide , 1795, 1796, 1803, 1804, 1808, 1810, 1864, 1903, 1911, 2091, 2118 and 2284, and preferably at one or more positions 377, 378, 468, 491, 504, 556 and 711, and the The conjugate has less degradation from plasma proteases that degrade Factor VIII than the unconjugated polypeptide. More preferably, the plasma protease is activated protein C.
在另一个实施方案中，生物相容的聚合物共价附着在B-结构域缺失的因子VIII的氨基酸位置129、491、1804和/或1808，更优选地，491或1808。在另一个实施方案中，生物相容的聚合物附着在多肽的因子VIII氨基酸位置1804，且包含聚乙二醇。优选地，生物相容的聚合物附着的一个或多个预定位点由位点特异性的半胱氨酸突变控制。In another embodiment, the biocompatible polymer is covalently attached at amino acid positions 129, 491, 1804 and/or 1808, more preferably, 491 or 1808, of B-domain deleted Factor VIII. In another embodiment, the biocompatible polymer is attached to the polypeptide at amino acid position 1804 of Factor VIII and comprises polyethylene glycol. Preferably, the one or more predetermined sites of attachment of the biocompatible polymer are controlled by site-specific cysteine mutations.
功能因子VIII多肽上的一个或多个(优选地，一个或两个)位点，可以是聚合物附着的预定位点。在具体的实施方案中，多肽是单-PEG化的或双PEG化的。One or more (preferably, one or two) sites on the functional Factor VIII polypeptide may be predetermined sites for polymer attachment. In specific embodiments, the polypeptide is mono-PEGylated or double-PEGylated.
本发明也涉及制备缀合物的方法，其包含：突变编码功能因子VIII多肽的核苷酸序列，以在预定位点用半胱氨酸残基的编码序列替代；表达突变的核苷酸序列，以产生半胱氨酸增强的突变蛋白；纯化该突变蛋白；使突变蛋白与生物相容的聚合物反应，所述聚合物已经被激活，以与多肽在基本上仅还原的半胱氨酸残基处反应，从而形成缀合物；和纯化该缀合物。在另一个实施方案中，本发明提供了位点定向PEG化因子VIII突变蛋白的方法，其包含：(a)表达位点定向因子VIII突变蛋白，其中所述突变蛋白在因子VIII突变蛋白的暴露表面上用半胱氨酸替代氨基酸残基，且该半胱氨酸被加帽；(b)在温和地还原半胱氨酸突变蛋白和释放帽的条件下，使半胱氨酸突变蛋白接触还原剂；(c)从半胱氨酸突变蛋白去除帽和还原剂；和(d)去除还原剂后至少约5分钟，且优选地，至少15分钟，更优选地，至少30分钟，在产生PEG化的因子VIII突变蛋白的条件下，用包含巯基偶联部分的PEG处理半胱氨酸突变蛋白。PEG的巯基偶联部分选自：硫羟、三氟甲磺酸、2，2，2-三氟乙磺酸、氮丙啶、环氧乙烷、S-吡啶基和马来酰亚胺部分，优选地，马来酰亚胺。The present invention also relates to a method for preparing a conjugate comprising: mutating the nucleotide sequence encoding a functional Factor VIII polypeptide to replace the coding sequence with a cysteine residue at a predetermined position; expressing the mutated nucleotide sequence , to produce a cysteine-enhanced mutein; purifying the mutein; reacting the mutein with a biocompatible polymer that has been activated to bind substantially only the reduced cysteine of the polypeptide reacting at the residue to form a conjugate; and purifying the conjugate. In another embodiment, the present invention provides a method for site-directed PEGylation of a Factor VIII mutein, comprising: (a) expressing a site-directed Factor VIII mutein, wherein said mutein is exposed upon exposure of the Factor VIII mutein Replacement of amino acid residues with cysteine on the surface, and the cysteine is capped; (b) contacting the cysteine mutein under conditions that gently reduce the cysteine mutein and release the cap reducing agent; (c) removing the cap and reducing agent from the cysteine mutein; and (d) at least about 5 minutes after removal of the reducing agent, and preferably, at least 15 minutes, more preferably, at least 30 minutes, after producing In the case of a PEGylated Factor VIII mutein, the cysteine mutein is treated with PEG containing a sulfhydryl coupling moiety. The thiol coupling moiety of PEG is selected from the group consisting of thiol, trifluoromethanesulfonic acid, 2,2,2-trifluoroethanesulfonic acid, aziridine, oxirane, S-pyridyl and maleimide moieties , preferably maleimide.
本发明还涉及肠胃外施用的药物组合物，其包含治疗有效量的本发明的缀合物和可药用佐剂。可药用佐剂是可以加入活性成分中来辅助配制或稳定化制剂且不会对患者造成显著的有害毒理学作用的物质。这样的佐剂的实例是本领域技术人员熟知的，且包括水、糖如麦芽糖或蔗糖、清蛋白、盐等。其它佐剂记载在例如E.W.Martin的Remington′sPharmaceutical Sciences中。这样的组合物将含有有效量的缀合物以及适宜量的载体，以制备适合有效地施用给宿主的可药用组合物。例如，缀合物可以肠胃外地施用给遭受血友病A的受试者，其剂量可以随出血发作的严重性而变化。静脉内施用的平均剂量范围是，对于手术前适应症是40单位/kg，对于微量出血是15-20单位/kg，且对于维持剂量是在8小时时间段内施用的20-40单位/kg。The present invention also relates to pharmaceutical compositions for parenteral administration comprising a therapeutically effective amount of a conjugate of the invention and a pharmaceutically acceptable adjuvant. A pharmaceutically acceptable adjuvant is a substance that can be added to the active ingredient to aid in the formulation or stabilize the formulation without causing significant adverse toxicological effects to the patient. Examples of such adjuvants are well known to those skilled in the art and include water, sugars such as maltose or sucrose, albumin, salts and the like. Other adjuvants are described, for example, in Remington's Pharmaceutical Sciences by E.W. Martin. Such compositions will contain an effective amount of the conjugate together with an appropriate amount of carrier to produce a pharmaceutically acceptable composition suitable for effective administration to a host. For example, the conjugate can be administered parenterally to a subject suffering from hemophilia A, and the dosage can vary with the severity of the bleeding episode. Average dose ranges for intravenous administration were 40 Units/kg for preoperative indications, 15-20 Units/kg for minor bleeding, and 20-40 Units/kg administered over an 8-hour period for maintenance doses .
在一个实施方案中，本发明的方法包含，用半胱氨酸替代一个或多个表面BDD氨基酸，在哺乳动物表达系统中生产半胱氨酸突变蛋白，还原在表达过程中已经被生长培养基中的半胱氨酸加帽的半胱氨酸，去除还原剂，以允许BDD二硫键重新形成，和与半胱氨酸-特异性的生物相容的聚合物试剂(如PEG-马来酰亚胺)反应。这样的试剂的实例是PEG-马来酰亚胺，其中PEG大小为如5、22或43kD，可以从Nektar Therapeutics of San Carlos，CA得到，Nektar目录号分别是2D2M0H01 mPEG-MAL MW 5,000Da、2D2M0P01 mPEG-MAL MW 20kD、2D3X0P01 mPEG2-MAL MW 40kD，或PEG大小为12或33kD，可以从NOF Corporation，Tokyo，日本得到，NOF目录号分别是Sunbright ME-120MA和Sunbright ME-300MA。使用离子交换层析，纯化PEG化的产物，以去除未反应的PEG，并使用尺寸排阻层析来去除未反应的BDD。该方法可以用于鉴别和选择性地屏蔽任何与FVIII的不利反应，如受体-介导的清除、抑制性抗体结合和蛋白水解酶的降解。我们注意到，Nektar或NOF提供的5kD PEG试剂在我们实验室测得为6kD，且类似地，作为线性20kD提供的PEG试剂在我们实验室测得为22kD，作为40kD提供的测得为43kD，且作为60kD提供的测得为64kD。为了避免混乱，在本文的讨论中，我们使用在我们实验室测得的分子量，例外是5kD PEG，我们将它记录为与生产商的鉴别相同的5kD。In one embodiment, the method of the invention comprises, replacing one or more surface BDD amino acids with cysteine, producing a cysteine mutein in a mammalian expression system, reducing the amount of cysteine mutein that has been removed from the growth medium during expression. Capping cysteines in cysteines removes the reducing agent to allow reformation of the BDD disulfide bond, and reacts with cysteine-specific biocompatible polymer reagents (such as PEG-malay imide) reaction. An example of such a reagent is PEG-maleimide, where the PEG size is e.g. 5, 22 or 43 kD, available from Nektar Therapeutics of San Carlos, CA, Nektar catalog numbers are 2D2M0H01 mPEG-MAL MW 5,000 Da, 2D2M0P01 respectively mPEG-MAL MW 20kD, 2D3X0P01 mPEG2-MAL MW 40kD, or PEG size 12 or 33kD, are available from NOF Corporation, Tokyo, Japan under NOF catalog numbers Sunbright ME-120MA and Sunbright ME-300MA, respectively. The PEGylated product was purified using ion exchange chromatography to remove unreacted PEG and size exclusion chromatography to remove unreacted BDD. This method can be used to identify and selectively mask any adverse reactions with FVIII, such as receptor-mediated clearance, inhibitory antibody binding and degradation by proteolytic enzymes. We note that the 5kD PEG reagents supplied by Nektar or NOF measured 6kD in our laboratory and similarly the PEG reagents supplied as linear 20kD measured 22kD in our laboratory and the ones supplied as 40kD measured 43kD, And that provided as 6OkD measured 64kD. To avoid confusion, in this discussion we use molecular weights measured in our laboratory, with the exception of 5kD PEG, which we report as the same 5kD as the manufacturer's identification.
除了在BDD的位置491和1808处的半胱氨酸突变(上面公开的)外，将位置487、496、504、468、1810、1812、1813、1815、1795、1796、1803和1804突变成半胱氨酸，以潜在地允许在PEG化之后阻断LRP结合。同样，将位置377、378和556突变成半胱氨酸，以允许在PEG化之后阻断LRP和HSPG结合。选择位置81、129、422、523、570、1864、1911、2091和2284以使其在BDD上相等地间隔开，从而使得在这些位置用大PEG(＞40kD)进行的位点定向PEG化以及在天然糖基化位点(41、239和2118)和LRP结合位点的PEG化完全覆盖BDD的表面，并鉴别BDD的新清除机理。In addition to the cysteine mutations at positions 491 and 1808 of the BDD (disclosed above), positions 487, 496, 504, 468, 1810, 1812, 1813, 1815, 1795, 1796, 1803 and 1804 were mutated to Cysteine to potentially allow blocking of LRP binding after PEGylation. Likewise, positions 377, 378 and 556 were mutated to cysteines to allow blocking of LRP and HSPG binding after PEGylation. Positions 81, 129, 422, 523, 570, 1864, 1911, 2091, and 2284 were chosen to be equally spaced across the BDD to allow site-directed PEGylation with large PEGs (>40kD) at these positions and PEGylation at native glycosylation sites (41, 239 and 2118) and the LRP binding site completely covered the surface of BDD and identified a new clearance mechanism for BDD.
在一个实施方案中，细胞培养基含有半胱氨酸，其通过形成二硫键，“加帽”突变蛋白上的半胱氨酸残基。在缀合物的制备中，在重组系统中生产的半胱氨酸突变蛋白被培养基中的半胱氨酸加帽，且该帽被释放帽的温和还原去除，然后添加半胱氨酸-特异性的聚合物试剂。如本领域技术人员会明白的，也可以使用本领域已知的FVIII位点特异性突变的其它方法。In one embodiment, the cell culture medium contains cysteine, which "caps" the cysteine residue on the mutein by forming a disulfide bond. In the preparation of the conjugate, the cysteine mutein produced in the recombinant system is capped with cysteine in the medium, and the cap is removed by mild reduction of the release cap, followed by addition of cysteine- Specific polymer reagents. Other methods of site-specific mutagenesis of FVIII known in the art may also be used, as will be apparent to those skilled in the art.
实施例Example
FVIII的结构活性关系分析。FVIII和BDD FVIII是非常大的复杂分子，具有许多不同的参与生物学反应的位点。以前共价修饰它们以提高药物代谢动力学性质的尝试具有混合的结果。令人惊奇的是，该分子可以被特异性地突变，然后以位点特异性的方式添加聚合物。此外，已知过去的聚合缀合物造成非特异性添加和降低的活性的问题，提高的药物代谢动力学性质和保留的活性的结果也令人惊奇。Structure-activity relationship analysis of FVIII. FVIII and BDD FVIII are very large complex molecules with many different sites involved in biological reactions. Previous attempts to covalently modify them to enhance pharmacokinetic properties have had mixed results. Surprisingly, the molecule could be specifically mutated and the polymer added in a site-specific manner. Furthermore, the improved pharmacokinetic properties and retained activity are also surprising results, given that past polymeric conjugates have caused problems with non-specific addition and reduced activity.
在一个实施方案中，本发明涉及使用半胱氨酸-特异性的配体(如PEG-马来酰亚胺)的位点定向诱变。未突变的BDD不具有任何可利用的与PEG-马来酰亚胺反应的半胱氨酸，所以仅仅突变的半胱氨酸位置将是PEG化位点。更具体地，BDD FVIII具有19个半胱氨酸，其中16个形成二硫键，且另外3个是游离的半胱氨酸(McMullen等，1995，ProteinSci.4，pp.740-746)。BDD的结构模型表明，所有3个游离半胱氨酸都是隐蔽的(Stoliova-McPhie等，2002，Blood 99，pp.1215-1223)。因为氧化的半胱氨酸不能被PEG-马来酰亚胺PEG化，所以如果不首先还原，BDD中形成二硫键的16个半胱氨酸就不能被PEG化。基于BDD的结构模型，如果不首先使蛋白变性，以将BDD中的3个游离的半胱氨酸暴露于PEG试剂，这些半胱氨酸就不能被PEG化。因而，似乎不可能通过在天然半胱氨酸残基处PEG化来实现BDD的特异性的PEG化，而不显著改变BDD结构，这最可能破坏它的功能。In one embodiment, the invention involves site-directed mutagenesis using cysteine-specific ligands such as PEG-maleimide. Unmutated BDD does not have any cysteines available to react with PEG-maleimide, so only mutated cysteine positions will be PEGylation sites. More specifically, BDD FVIII has 19 cysteines, of which 16 form disulfide bonds and the other 3 are free cysteines (McMullen et al., 1995, Protein Sci. 4, pp. 740-746). Structural models of BDD show that all 3 free cysteines are cryptic (Stoliova-McPhie et al., 2002, Blood 99, pp. 1215-1223). Because oxidized cysteines cannot be PEGylated by PEG-maleimide, the 16 cysteines in BDD that form disulfide bonds cannot be PEGylated without first reducing them. Based on the structural model of BDD, the 3 free cysteines in BDD cannot be PEGylated without first denaturing the protein to expose these cysteines to the PEG reagent. Thus, it appears impossible to achieve specific PEGylation of BDD by PEGylation at native cysteine residues without significantly altering the BDD structure, most likely disrupting its function.
不清楚全长FVIII的B结构域中的4个半胱氨酸的氧化还原状态。B结构域中的4个半胱氨酸如果不形成二硫键且暴露于表面，它们就可能被PEG化。但是，因为全长FVIII和BDD具有类似的药物代谢动力学(PK)特征和类似的体内半衰期(Gruppo等，2003，Haemophilia 9，pp.251-260)，B结构域PEG化不太可能导致提高的血浆半衰期，除非PEG的发生也保护非-B结构域区域。The redox state of the four cysteines in the B domain of full-length FVIII is unclear. The 4 cysteines in the B domain are likely to be PEGylated if they do not form disulfide bonds and are surface exposed. However, because full-length FVIII and BDD have similar pharmacokinetic (PK) profiles and similar half-lives in vivo (Gruppo et al., 2003, Haemophilia 9, pp.251-260), it is unlikely that PEGylation of the B domain results in increased plasma half-life unless PEG occurs that also protects the non-B domain region.
为了确定具有FVIII活性的多肽上用于聚合物附着的预定位点，所述附着将保留因子VIII活性和提高药物代谢动力学，基于BDD FVIII提出了下面的指导。修饰应当靶向清除、灭活和免疫原性机理，如LRP、HSPG、APC和抑制性抗体结合位点。Stoilova-McPhie.S.等，2002，Blood 99(4)，pp.1215-23显示了BDD的结构。例如，为了延长半衰期，可以将单个PEG导入在A2残基484-509和A3残基1811-1818中的LRP结合位点处或附近的特异性位点。在这些位点导入大体积的PEG，会破坏FVIII结合LRP的能力，并减少FVIII从循环中的清除。还认为，为了延长半衰期而不显著影响活性，可以将PEG导入残基1648，它位于全长分子中的B结构域和A3结构域的接点和A2和A3结构域之间的BDD的14-氨基酸接头中。In order to identify predetermined sites on polypeptides with FVIII activity for polymer attachment that would preserve Factor VIII activity and improve pharmacokinetics, the following guidance was proposed based on BDD FVIII. Modifications should target clearance, inactivation and immunogenicity mechanisms such as LRP, HSPG, APC and inhibitory antibody binding sites. Stoilova-McPhie.S. et al., 2002, Blood 99(4), pp.1215-23 show the structure of BDD. For example, to increase half-life, a single PEG can be introduced into specific sites at or near the LRP binding site in A2 residues 484-509 and A3 residues 1811-1818. Introduction of bulky PEG at these sites disrupts the ability of FVIII to bind LRP and reduces clearance of FVIII from circulation. It is also believed that in order to prolong half-life without significantly affecting activity, PEG could be introduced at residue 1648, which is located at the junction of the B and A3 domains and the 14-amino acid of the BDD between the A2 and A3 domains in the full-length molecule in the connector.
通过使用重组DNA诱变技术，将单个半胱氨酸残基工程改造进A2或A3结构域，随后用半胱氨酸-特异性的PEG试剂(如PEG-马来酰亚胺)位点特异性地PEG化导入的半胱氨酸，可以实现PEG化的特异性。在484-509和1811-1818处PEG化的另一个优点是，这两个表位代表着患者的2/3大类抑制性抗原位点。为了达到提高的循环半衰期和免疫原性反应的减少的最大效果，可以将A2和A3 LRP结合位点两者PEG化，以产生双PEG化的产物。应当指出，在1811-1818区域内PEG化，可导致活性的显著损失，因为该区域也参与FIX结合。558-565内的位点定向PEG化应当废除HSPG结合，但是也可减少活性，因为该区域也结合FIX。By using recombinant DNA mutagenesis techniques, single cysteine residues are engineered into the A2 or A3 domains, followed by site-specific cysteine-specific PEG reagents (eg, PEG-maleimide) The specificity of PEGylation can be achieved by selectively PEGylating the introduced cysteine. Another advantage of PEGylation at 484-509 and 1811-1818 is that these two epitopes represent 2/3 of the largest class of inhibitory antigenic sites in patients. To achieve the maximal effect of increased circulating half-life and reduction of immunogenic responses, both the A2 and A3 LRP binding sites can be PEGylated to generate a double PEGylated product. It should be noted that PEGylation in the region 1811-1818 can lead to a significant loss of activity since this region is also involved in FIX binding. Site-directed PEGylation within 558-565 should abrogate HSPG binding, but could also reduce activity since this region also binds FIX.
可以PEG化其它表面位点，以鉴别新的FVIII清除机理。A2结构域的PEG化可以提供另外的优点，因为A2结构域在激活后会从FVIII解离，且推测由于它更小的大小，它可以比FVIII分子剩余部分更快地从循环中去除。另一方面，PEG化的A2可以大得足以逃避肾清除，且具有与FVIII剩余部分相当的血浆半衰期，从而可以体内重建激活的FVIII。Other surface sites can be PEGylated to identify novel mechanisms of FVIII clearance. PEGylation of the A2 domain may provide additional advantages, as the A2 domain dissociates from FVIII upon activation and presumably due to its smaller size it may be removed from circulation faster than the rest of the FVIII molecule. On the other hand, PEGylated A2 can be large enough to escape renal clearance and have a comparable plasma half-life to the rest of FVIII, allowing reconstitution of activated FVIII in vivo.
A2和A3区域中的PEG化位点的鉴别。基于高表面暴露和它们的Ca向Cβ轨迹的向外方向，选择在推定的A2 LRP结合区域处或附近的5个位置(与PEG1-5位置相对应的Y487、L491、K496、L504和Q468)，作为位点定向PEG化的实例。此外，这些残基在分子的三维结构中彼此大致等距，从而它们一起可以代表该整个区域。选择在推定的A3LRP结合区域处或附近的8个位置(与PEG6-14相对应的1808、1810、1812、1813、1815、1795、1796、1803、1804)，作为位点定向PEG化的实例。PEG6(K1808)邻近1811-1818和在1810处的天然N-连接的糖基化位点。在位置1810处的PEG化(PEG7)将用PEG替代糖。在PEG8位置T1812处的突变，也将废除糖基化位点。尽管预测PEG9位置(K1813)指向内侧，但在结构模型不正确的情况下选择它。PEG10(Y1815)是在LRP结合环内的大体积的疏水氨基酸，且可以是关键的相互作用残基，因为疏水氨基酸通常存在于蛋白-蛋白相互作用的中心。因为已经报道1811-1818区域会参与LRP和FIX结合两者，所以认为该环内的PEG化可能导致降低的活性。因而，PEG11-PEG14(1795、1796、1803、1804)被设计成在1811-1818环附近，但是不在环内，从而人们可以用不同的PEG大小解离LRP和FIX结合。Identification of PEGylation sites in the A2 and A3 regions. Five positions (Y487, L491, K496, L504 and Q468 corresponding to PEG1-5 positions) at or near the putative A2 LRP binding region were selected based on high surface exposure and outward orientation of their Ca towards the Cβ track , as an example of site-directed PEGylation. Furthermore, these residues are approximately equidistant from each other in the three-dimensional structure of the molecule so that together they can represent the entire region. Eight positions (1808, 1810, 1812, 1813, 1815, 1795, 1796, 1803, 1804 corresponding to PEG6-14) at or near the putative A3LRP binding region were chosen as examples of site-directed PEGylation. PEG6(K1808) is adjacent to 1811-1818 and the native N-linked glycosylation site at 1810. PEGylation (PEG7) at position 1810 will replace the sugar with PEG. A mutation at position T1812 of PEG8 will also abolish the glycosylation site. Although the PEG9 position (K1813) was predicted to point medially, it was chosen in the case of an incorrect structural model. PEG10 (Y1815) is a bulky hydrophobic amino acid within the LRP binding loop and may be a key interacting residue since hydrophobic amino acids are often found at the center of protein-protein interactions. Since the 1811-1818 region has been reported to be involved in both LRP and FIX binding, it was thought that PEGylation within this loop might result in reduced activity. Thus, PEG11-PEG14 (1795, 1796, 1803, 1804) were designed near the 1811-1818 loop, but not within the loop, so that one can dissociate LRP and FIX binding with different PEG sizes.
为了同时阻断两个LRP结合位点，可以生成双PEG化，例如，在PEG2和PEG6位置。In order to block both LRP binding sites simultaneously, double PEGylation can be generated, for example, at the PEG2 and PEG6 positions.
因为已经显示558-565区域结合HSPG和FIX两者，在该区域内没有设计位点。相反地，将PEG15-PEG17(377、378和556)设计在A2LRP和HSPG结合区域之间，从而使得附着的PEG可以干扰它们之间的相互作用并破坏可能的相互作用。也可以选择在LRP和HPSG结合区域内或附近的表面暴露的且指向外侧的其它位点。为了鉴别新的清除机理，可以将FVIII系统地PEG化。除了PEG1-17以外，3个其它的天然糖基化位点，即与PEG18-20相对应的N41、N239和N2118，可以用作PEG化的限定点(tethering point)，因为它们应当是表面暴露的。除了vWF、FIX、FX、磷脂和凝血酶的功能性相互作用位点以外，将在离PEG2、PEG6的Cβ原子20埃半径内的表面积和4个糖基化位点作图到BDD模型上。Since the region 558-565 has been shown to bind both HSPG and FIX, no sites were designed within this region. Conversely, PEG15-PEG17 (377, 378, and 556) were engineered between the A2LRP and HSPG-binding regions such that the attached PEG could interfere with their interactions and disrupt possible interactions. Other sites that are surface exposed and point outward in or near the LRP and HPSG binding region may also be selected. To identify novel clearance mechanisms, FVIII can be systematically PEGylated. In addition to PEG1-17, three other natural glycosylation sites, namely N41, N239 and N2118 corresponding to PEG18-20, can be used as tethering points for PEGylation since they should be surface exposed of. In addition to the functional interaction sites of vWF, FIX, FX, phospholipids and thrombin, the surface area within a 20 angstrom radius from the Cβ atom of PEG2, PEG6 and 4 glycosylation sites were mapped onto the BDD model.
然后，基于它们覆盖离它们每个Cβ原子20埃半径的几乎整个剩余BDD表面的能力，选择与Y81、F129、K422、K523、K570、N1864、T1911、Q2091和Q2284相对应的PEG21-29。还选择这些位置，因为它们是完全暴露的，指向外侧的，且远离天然半胱氨酸，以使可能的不正确的二硫键形成最小化。选择20埃半径，因为预期大PEG(如64kD分支的PEG)具有覆盖约20埃半径的球的潜力。一起PEG化PEG21-29以及PEG2和PEG6和糖基化位点PEG18、19和20，可能保护几乎整个无功能的FVIII表面。Then, PEG21-29 corresponding to Y81, F129, K422, K523, K570, N1864, T1911, Q2091 and Q2284 were selected based on their ability to cover almost the entire remaining BDD surface at a radius of 20 Angstroms from each of their Cβ atoms. These positions were also chosen because they are fully exposed, pointing outward, and away from native cysteines to minimize possible incorrect disulfide bond formation. The 20 Angstrom radius was chosen because large PEGs, such as 64kD branched PEGs, are expected to have the potential to cover spheres with a radius of about 20 Angstroms. PEGylation of PEG21-29 together with PEG2 and PEG6 and glycosylation sites PEG18, 19 and 20 together, likely protects almost the entire non-functional FVIII surface.
可以组合导致增强的性质(如提高的PK特征、更大的稳定性或减少的免疫原性)的PEG化位置，以生成具有最大增强性质的多-PEG化产物。通过分别去除A2和A3结构域中暴露的二硫键，设计PEG30和PEG31。PEG30或C630A应释放它的二硫键配偶体C711用于PEG化。同样地，PEG31，C1899A应允许C1903被PEG化。PEGylation positions that result in enhanced properties (eg, improved PK profile, greater stability, or reduced immunogenicity) can be combined to generate multi-PEGylated products with maximally enhanced properties. PEG30 and PEG31 were designed by removing the exposed disulfide bonds in the A2 and A3 domains, respectively. PEG30 or C630A should release its disulfide partner C711 for PEGylation. Likewise, PEG31, C1899A should allow C1903 to be PEGylated.
诱变。通过在选择用于PEG化的位点导入半胱氨酸密码子，可以生成FVIII的位点定向PEG化的底物。Stratagene cQuickChangeTM II位点定向诱变试剂盒用于制备所有的PEG突变体(Stratagene试剂盒200523，购自Stratagene Corporation，La JoIIa1 CA)。使用DNA聚合酶和温度循环仪，进行cQuikChangeTM位点定向诱变方法。使用不置换引物的PfuTurbo延伸两种互补的寡核苷酸引物(其中含有所需的突变)。使用含有野生型FVIII基因的dsDNA作为模板。在多个延伸循环后，用对甲基化的DNA特异性的DpnI内切核酸酶消化产物。新合成的含有突变的DNA是未甲基化的，而亲本野生型DNA是甲基化的。然后，将消化的DNA用于转化XL-1 Blue超感受态细胞。mutagenesis. Substrates for site-directed PEGylation of FVIII can be generated by introducing cysteine codons at sites selected for PEGylation. Stratagene cQuickChange™ II site-directed mutagenesis kit was used to prepare all PEG mutants (Stratagene kit 200523, purchased from Stratagene Corporation, La JoIIa1 CA). use DNA polymerase and temperature cycler for the cQuikChange™ site-directed mutagenesis method. Two complementary oligonucleotide primers (containing the desired mutations) were extended using PfuTurbo without primer displacement. dsDNA containing the wild-type FVIII gene was used as template. After multiple extension cycles, the product was digested with the DpnI endonuclease specific for methylated DNA. The newly synthesized DNA containing the mutation is unmethylated, whereas the parental wild-type DNA is methylated. Then, the digested DNA was used to transform XL-1 Blue supercompetent cells.
诱变效率是几乎80％。在pSK207+BDD C2.6或pSK207+BDD中进行诱变反应(图1)。通过DNA测序证实成功的诱变，并将含有突变的适当片段转入哺乳动物表达载体pSS207+BDD的FVIII主链中。转移后，将所有突变再次确认序列。对于A3突变蛋白PEG6、7、8、9和10，在载体pSK207+BDD C2.6中进行诱变。通过测序证实后，将突变体片段KpnI/Pme亚克隆进pSK207+BDD中。然后，将BDD突变蛋白亚克隆进pSS207+BDD表达载体中。对于A3突变蛋白PEG11、12、13、14，直接在载体pSK207+BDD中进行诱变，且然后将确认序列的突变体BDD亚克隆进pSS207+BDD中。对于A2突变蛋白PEG1、2、3、4、5，在pSK207+BDD C2.6载体中进行诱变。将确认序列的突变体亚克隆进pSK207+BDD中，且然后亚克隆进pSS207+BDD中。Mutagenesis efficiency was almost 80%. Mutagenesis reactions were performed in pSK207+BDD C2.6 or pSK207+BDD (Figure 1). Successful mutagenesis was confirmed by DNA sequencing, and the appropriate fragment containing the mutation was transferred into the FVIII backbone of the mammalian expression vector pSS207+BDD. After transfer, all mutations were re-confirmed for sequence. For the A3 muteins PEG6, 7, 8, 9 and 10, mutagenesis was performed in the vector pSK207+BDD C2.6. After confirmation by sequencing, the mutant fragment KpnI/Pme was subcloned into pSK207+BDD. Then, the BDD mutein was subcloned into the pSS207+BDD expression vector. For the A3 muteins PEG11, 12, 13, 14, mutagenesis was performed directly in the vector pSK207+BDD, and then the sequence-confirmed mutant BDD was subcloned into pSS207+BDD. For the A2 muteins PEG1, 2, 3, 4, 5, mutagenesis was performed in the pSK207+BDD C2.6 vector. Sequence confirmed mutants were subcloned into pSK207+BDD and then into pSS207+BDD.
列出了每个反应的诱变所使用的引物(仅有义链)：Primers used for mutagenesis (sense strand only) for each reaction are listed:
PEG1，Y487C：GATGTCCGTCCTTTGTGCTCAAGGAGATTACCA(SEQ ID NO：5)PEG1, Y487C: GATGTCCGTCCTTTGTGCTCAAGGAGATTACCA (SEQ ID NO: 5)
PEG2，L491C：TTGTATTCAAGGAGATGCCCAAAAGGTGTAAAAC(SEQ ID NO：6)PEG2, L491C: TTGTATTCAAGGAGATGCCCAAAAGGTGTAAAAC (SEQ ID NO: 6)
PEG3，K496c：TTACCAAAAGGTGTATGCCATTTGAAGGATTTTC(SEQ ID NO：7)PEG3, K496c:TTACCAAAAGGTGTATGCCATTTGAAGGATTTTC (SEQ ID NO: 7)
PEG4，L504C：AAGGATTTTCCAATTTGCCCAGGAGAAATATTC(SEQ ID NO：8)PEG4, L504C: AAGGATTTTCCAATTTGCCCAGGAGAAATATTC (SEQ ID NO: 8)
PEG5，Q468C：GATTATATTTAAGAATTGCGCAAGCAGACCATAT(SEQ ID NO：9)PEG5, Q468C: GATTATATTTAAGAATTGCGCAAGCAGACCATAT (SEQ ID NO: 9)
PEG6，K1808C：TAGAAAAAACTTTGTCTGCCCTAATGAAACCAAAAC(SEQ ID NO：10)PEG6, K1808C: TAGAAAAAACTTTGTCTGCCCTAATGAAACCAAAAC (SEQ ID NO: 10)
PEG7，N1810C：AACTTTGTCAAGCCTTGCGAAACCAAAACTTAC(SEQ ID NO：11)PEG7, N1810C: AACTTTGTCAAGCCTTGCGAAACCAAAACTTAC (SEQ ID NO: 11)
PEG8，T1812C：GTCAAGCCTAATGAATGCAAAACTTACTTTTGGA(SEQ ID NO：12)PEG8, T1812C: GTCAAGCCTAATGAATGCAAAACTTACTTTTGGA (SEQ ID NO: 12)
PEG9，K1813C：CAAGCCTAATGAAACCTGCACTTACTTTTGGAAAG(SEQ ID NO：13)PEG9, K1813C: CAAGCCTAATGAAACCTGCACTTACTTTTGGAAAG (SEQ ID NO: 13)
PEG10，Y11815C：CTAATGAAACCAAAACTTGCTTTTGGAAAGTGCAAC(SEQ ID NO：14)PEG10, Y11815C: CTAATGAAACCAAAACTTGCTTTTGGAAAGTGCAAC (SEQ ID NO: 14)
PEG11，D1795C：ATTTCTTATGAGGAATGCCAGAGGCAAGGAGCA(SEQ ID NO：15)PEG11, D1795C: ATTTCTTATGAGGAATGCCAGAGGCAAGGAGCA (SEQ ID NO: 15)
PEG12，Q1796C：TCTTATGAGGAAGATTGCAGGCAAGGAGCAGAA(SEQ ID NO：16)PEG12, Q1796C: TCTTATGAGGAAGATTGCAGGCAAGGAGCAGAA (SEQ ID NO: 16)
PEG13，R1803C：CAAGGAGCAGAACCTTGCAAAAACTTTGTCAAGCCT(SEQ ID NO：17)PEG13, R1803C: CAAGGAGCAGAACCTTGCAAAAACTTTGTCAAGCCT (SEQ ID NO: 17)
PEG14，K1804C：GGAGCAGAACCTAGATGCAACTTTGTCAAGCCT(SEQ ID NO：18)PEG14, K1804C: GGAGCAGAACCTAGATGCAACTTTGTCAAGCCT (SEQ ID NO: 18)
PEG15，K377C：CGCTCAGTTGCCAAGTGTCATCCTAAAACTTGG(SEQ ID NO：19)PEG15, K377C: CGCTCAGTTGCCAAGTGTCATCCTAAAACTTGG (SEQ ID NO: 19)
PEG16，H378C：TCAGTTGCCAAGAAGTGTCCTAAAACTTGGGTA(SEQ ID NO：20)PEG16, H378C: TCAGTTGCCAAGAAGTGTCCTAAAACTTGGGTA (SEQ ID NO: 20)
PEG 17，K556C：CTCCTCATCTGCTACTGCGAATCTGTAGATCAA(SEQ ID NO：21)PEG 17, K556C: CTCCTCATCTGCTACTGCGAATCTGTAGATCAA (SEQ ID NO: 21)
PEG18，N41C：CAAAATCTTTTCCATTCTGCACCTCAGTCGTGTAC(SEQ ID NO：22）PEG18, N41C: CAAAATCTTTTCCATTCTGCACCTCAGTCGTGTAC (SEQ ID NO: 22)
PEG19，N239C：GTCAATGGTTATGTATGCAGGTCTCTGCCAGGT(SEQ ID NO：23)PEG19, N239C: GTCAATGGTTATGTATGCAGGTCTCTGCCAGGT (SEQ ID NO: 23)
PEG20，N2118C：CAGACTTATCGAGGATGTTCCACTGGAACCTTA(SEQ ID NO：24)PEG20, N2118C: CAGACTTATCGAGGATGTTCCACTGGAACCTTA (SEQ ID NO: 24)
PEG21，Y81C：ATCCAGGCTGAGGTTTGTGATACAGTGGTCATT(SEQ ID NO：25)PEG21, Y81C: ATCCAGGCTGAGGTTTGTGATACAGTGGTCATT (SEQ ID NO: 25)
PEG22，F129C：GAAGATGATAAAGTCTGTCCTGGTGGAAGCCAT(SEQ ID NO：26)PEG22, F129C: GAAGATGATAAAGTCTGTCCTGGTGGAAGCCAT (SEQ ID NO: 26)
PEG23，K422C：CAGCGGATTGGTAGGTGTTACAAAAAAGTCCGA(SEQ ID NO：27)PEG23, K422C: CAGCGGATTGGTAGGTGTTACAAAAAGTCCGA (SEQ ID NO: 27)
PEG24，K523C：GAAGATGGGCCAACTTGCTCAGATCCTCGGTGC(SEQ ID NO：28)PEG24, K523C: GAAGATGGGCCAACTTGCTCAGATCCTCGGTGC (SEQ ID NO: 28)
PEG25，K570C：CAGATAATGTCAGACTGCAGGAATGTCATCCTG(SEQ ID NO：29)PEG25, K570C: CAGATAATGTCAGACTGCAGGAATGTCATCCTG (SEQ ID NO: 29)
PEG26，N1864C：CACACTAACACACTGTGTCCTGCTCATGGGAGA(SEQ ID NO：30)PEG26, N1864C: CACACTAACACACTGTGTCCTGCTCATGGGAGA (SEQ ID NO: 30)
PEG27，T1911C，CAGATGGAAGATCCCTGCTTTAAAGAGAATTAT(SEQ ID NO：31)PEG27, T1911C, CAGATGGAAGATCCCTGCTTTAAAGAGAATTAT (SEQ ID NO: 31)
PEG28，Q2091C：ACCCAGGGTGCCCGTTGCAAGTTCTCCAGCCTC(SEQ ID NO：32)PEG28, Q2091C: ACCCAGGGTGCCCGTTGCAAGTTCTCCAGCCTC (SEQ ID NO: 32)
PEG29，Q2284C：AAAGTAAAGGTTTTTTGCGGAAATCAAGACTCC(SEQ ID NO：33)PEG29, Q2284C: AAAGTAAAGGTTTTTTGCGGAAATCAAGACTCC (SEQ ID NO: 33)
PEG30，C630A：TTGCAGTTGTCAGTTGCTTTGCATGAGGTGGCA(SEQ ID NO：34)PEG30, C630A: TTGCAGTTGTCAGTTGCTTTGCATGAGGTGGCA (SEQ ID NO: 34)
PEG31，C1899A：AATATGGAAAGAAACGCTAGGGCTCCCTGCAAT(SEQ ID NO：35)PEG31, C1899A: AATATGGAAAGAAACGCTAGGGCTCCCTGCAAT (SEQ ID NO: 35)
突变蛋白表达。在插入赋予对潮霉素B抗性的载体后，按照生产商的说明书，将PEG突变蛋白转染进与293Fectin转染试剂(Invitrogen Corp.目录号12347-019)复合的HKB11细胞(美国专利6,136,599)中。通过Coatest生色测定(Chromogenix Corp.目录号821033，见实施例12生色测定)，评价转染后3天的FVIII表达(表1)。然后，在补加了5％FBS的生长培养基中，将转染的细胞置于50μg/ml的潮霉素B选择压力下。当出现潮霉素B抗性的菌落时，手工挑取它们，并通过Coatest生色测定筛选FVIII表达。然后，将FVIII表达稳定的细胞适应到含有HPPS补料的培养基中。扩增细胞，并以1X 106细胞/ml接种进含有新鲜培养基的摇瓶中。3天后收获的组织培养液(TCF)用于纯化FVIII BDD突变蛋白。通过Coatest测定TCF的FVIII活性(表1)。Mutant protein expression. After insertion of a vector conferring resistance to hygromycin B, the PEG mutein was transfected into HKB11 cells complexed with 293Fectin transfection reagent (Invitrogen Corp. Cat. No. 12347-019) according to the manufacturer's instructions (US Patent 6,136,599 )middle. FVIII expression was assessed 3 days after transfection by the Coatest chromogenic assay (Chromogenix Corp. Cat. No. 821033, see Example 12 Chromogenic Assay) (Table 1). Transfected cells were then placed under a selection pressure of 50 [mu]g/ml hygromycin B in growth medium supplemented with 5% FBS. When hygromycin B-resistant colonies emerged, they were manually picked and screened for FVIII expression by the Coatest chromogenic assay. Cells with stable FVIII expression were then adapted to medium containing HPPS feed. Cells were expanded and seeded into shake flasks containing fresh medium at 1X106 cells/ml. Tissue culture fluid (TCF) harvested after 3 days was used to purify the FVIII BDD mutein. The FVIII activity of TCF was determined by Coatest (Table 1).
PEG突变蛋白效价的总结Summary of PEG Mutant Protein Potency
表1.来自瞬时的和稳定的转染的PEG突变蛋白的表达水平Table 1. Expression levels of PEG muteins from transient and stable transfections
突变蛋白纯化。收集含有分泌的突变蛋白FVIII蛋白的细胞培养上清液后，通过0.2微米薄膜滤器过滤上清液，去除任何残存细胞。然后，通过超滤或阴离子交换，浓缩上清液。然后，将其应用于免疫亲和柱，在这里去除细胞培养基组分和大部分宿主细胞蛋白杂质。然后通过渗滤，将免疫亲和柱洗脱物缓冲液更换进含有蔗糖的配制缓冲液中，并冷冻。通过生色测定，评定经过单克隆FVIII抗体柱的蛋白得率和回收。测定层析运行的装载、流通(flow through)、各种洗脱物级分、带和渗滤的洗脱物样品的FVIII活性(表2)。表2显示了PEG2突变蛋白从单克隆抗体柱的回收。抗体是C7F7抗体。通过生色测定确定表2的百分比回收。终得率是73％。在图2中显示了经单克隆FVIII抗体层析柱纯化的PEG2蛋白的280nmUV吸光度关于时间的图。使用来自Amersham Bioscience的Explorer 100层析系统，进行层析。该装置采用多波长UV-可见光监控器和2mm流动池。在有高盐存在下，从柱洗脱PEG2突变蛋白，且通过280nm吸光度和FVIII活性测定，指示洗脱峰。Mutant protein purification. After collecting the cell culture supernatant containing the secreted mutant FVIII protein, filter the supernatant through a 0.2 micron membrane filter to remove any remaining cells. Then, the supernatant is concentrated by ultrafiltration or anion exchange. It is then applied to an immunoaffinity column where cell culture medium components and most host cell protein impurities are removed. The immunoaffinity column eluate was then buffer exchanged into formulation buffer containing sucrose by diafiltration and frozen. Protein yield and recovery over the monoclonal FVIII antibody column was assessed by chromogenic assay. Loading, flow through, various eluate fractions, bands and diafiltered eluate samples of the chromatography runs were assayed for FVIII activity (Table 2). Table 2 shows the recovery of PEG2 muteins from monoclonal antibody columns. Antibody is C7F7 antibody. The percent recovery of Table 2 was determined by chromogenic assay. The final yield was 73%. In FIG. 2 , the 280 nm UV absorbance of the PEG2 protein purified by monoclonal FVIII antibody chromatography column versus time is shown. Using from Amersham Bioscience Explorer 100 chromatography system, for chromatography. The device employs a multi-wavelength UV-visible light monitor and a 2mm flow cell. The PEG2 mutein was eluted from the column in the presence of high salt and the elution peak was indicated by absorbance at 280 nm and FVIII activity measurements.
表2.来自单克隆FVIII抗体柱的PEG2突变蛋白的回收Table 2. Recovery of PEG2 muteins from monoclonal FVIII antibody columns
PEG化。不经还原和在超过100倍过量的PEG：蛋白比(未显示数据)变性，半胱氨酸-特异性的PEG不能PEG化天然全长FVIII或BDD，从而证实了下述基于BDD结构模型的假说，即所有天然半胱氨酸都形成二硫键，或隐蔽在FVIII内。使用上述标准方案表达和纯化的FVIII半胱氨酸突变蛋白不能被半胱氨酸-特异性的PEG马来酰亚胺试剂PEG化，推测是因为导入的FVIII半胱氨酸通过与存在于细胞生长培养基中的巯基(如半胱氨酸和β-巯基乙醇)反应而被“加帽”。通过从培养基清除半胱氨酸和β-巯基乙醇，可以潜在地解决该问题，但是这可能导致更低的FVIII生成，且不能预防细胞释放的巯基封闭导入的FVIII半胱氨酸。PEGylation. Without reduction and denaturation at over 100-fold excess PEG:protein ratios (data not shown), cysteine-specific PEG was unable to PEGylate native full-length FVIII or BDD, thus confirming the following based on the BDD structural model Hypotheses, that all natural cysteines form disulfide bonds, or are sequestered within FVIII. FVIII cysteine muteins expressed and purified using the standard protocol described above cannot be PEGylated by cysteine-specific PEG-maleimide reagents, presumably because the introduced FVIII cysteines interact with cells present in the cells. Sulfhydryl groups (such as cysteine and β-mercaptoethanol) are reacted and "capped" in the growth medium. This problem could potentially be resolved by removing cysteine and β-mercaptoethanol from the medium, but this would lead to lower FVIII production and would not prevent the sulfhydryl groups released from the cells from blocking the imported FVIII cysteines.
在本发明的另一个方面，开发了三步方法，以允许FVIII的位点-特异性的PEG化(图3)。在步骤1中，用还原剂如约0.7mM三(2-羧乙基)膦(TCEP)或0.07mM二硫苏糖醇(DTT)，在4℃温和地还原约1μM的纯化的FVIII半胱氨酸突变蛋白30分钟，以释放“帽”。在步骤2中，通过尺寸排阻层析(SEC)方法，如使样品运行经过旋转柱以允许FVIII二硫键重新形成，同时保持导入的半胱氨酸游离并还原，与“帽”一起去除还原剂。在步骤3中，去除还原剂后至少30分钟，用至少10倍摩尔过量的大小范围为5-64kD的PEG-马来酰亚胺(Nektar Therapeutics和N.O.F.Corporation)在4℃处理游离的FVIII半胱氨酸突变蛋白至少1小时。该方法产生高度一致的产物谱，其对于由不同个体重复的许多反应，具有可再现的数据。In another aspect of the invention, a three-step method was developed to allow site-specific PEGylation of FVIII (Figure 3). In step 1, approximately 1 μM of purified FVIII cysteine is gently reduced at 4 °C with a reducing agent such as approximately 0.7 mM tris(2-carboxyethyl)phosphine (TCEP) or 0.07 mM dithiothreitol (DTT) Acid muteins for 30 minutes to release the "caps". In step 2, by a size exclusion chromatography (SEC) method such as running the sample through a spin column The reducing agent is removed along with the "cap" to allow reformation of the FVIII disulfide bond while leaving the introduced cysteine free and reduced. In step 3, at least 30 min after removal of the reducing agent, free FVIII cysteine was treated with at least a 10-fold molar excess of PEG-maleimide (Nektar Therapeutics and NOF Corporation) in the size range 5-64 kD at 4 °C Acid muteins for at least 1 hr. This method yielded highly consistent product profiles with reproducible data for many reactions repeated by different individuals.
因为去除TCEP的旋转柱方法不能改变比例，所以选择凝胶过滤脱盐层析。但是，在使用TCEP掺加样品(spike sample)测试该方法后，显示在柱空隙且不仅仅在盐级分中TCEP以可测量的水平洗脱，正如从具有小分子量的分子所预见到的。蛋白印迹测定显示显著的背景PEG化可能是由于TCEP的不完全去除。同时，分开的实验显示，使用与盐梯度相结合的阴离子交换层析介质，可以从其它蛋白杂质显著地进一步纯化C7F7纯化的材料。然后，决定用如上所述的TCEP还原C7F7材料，然后经阴离子交换柱加工该材料。因为电荷差异，FVIII蛋白会被保留，而TCEP会流过柱，且不会被保留。同时，在梯度盐洗脱的过程中，可以从大部分剩余蛋白杂质中纯化出FVIII蛋白。这意味着，以后发生的PEG化在理论上由于更纯的原材料而更均匀。但是，用TCEP的掺加样品测试后，发现可测量水平的TCEP洗脱在含有FVIII的梯度中。因此，决定在阴离子交换层析后执行凝胶过滤脱盐层析，从而当依次使用这两个步骤时，导致完全去除TCEP，并消除非特异性的PEG化。Gel filtration desalting chromatography was chosen because the spin-column method of removing TCEP cannot change the ratio. However, after testing the method using TCEP spike samples, it was shown that TCEP elutes at measurable levels in the column void and not only in the salt fraction, as expected from molecules with small molecular weights. Western blot assays showed significant background PEGylation likely due to incomplete removal of TCEP. At the same time, separate experiments showed that the C7F7 purified material could be significantly further purified from other protein impurities using anion exchange chromatography media combined with a salt gradient. It was then decided to reduce the C7F7 material with TCEP as described above and then process the material through an anion exchange column. Because of the difference in charge, the FVIII protein will be retained, while TCEP will flow through the column without being retained. At the same time, FVIII protein can be purified from most of the remaining protein impurities during gradient salt elution. This means that the PEGylation that occurs later is theoretically more homogeneous due to purer starting materials. However, upon testing with spiked samples of TCEP, it was found that measurable levels of TCEP eluted in the FVIII-containing gradient. Therefore, it was decided to perform gel filtration desalting chromatography after anion exchange chromatography, resulting in complete removal of TCEP and elimination of non-specific PEGylation when these two steps were used sequentially.
通过SDS PAGE和蛋白印迹进行PEG化分析。通过还原6％Tris甘氨酸SDS聚丙烯酰胺凝胶(Invitrogen)上的电泳，可以分析PEG化产物。电泳后，可以用考马斯蓝染色凝胶以鉴别所有蛋白，或进行标准的蛋白印迹规程，以鉴别FVIII的不同区域上的PEG化模式。用分别针对FVIII重链C-末端区域或VIII轻链N-末端区域产生的小鼠单克隆R8B12或C7F7抗体对印迹染色，应鉴别各链的PEG化。用针对FVIII的484-509区域的413抗体染色，将确定PEG化对于突变蛋白如PEG1-4是否确实是位点-特异性的。同样地，用识别FVIII的1801-1823区域的CLB-CAg A抗体染色，将确定PEG化对于突变蛋白如PEG6-10是否确实是位点-特异性的。PEGylation analysis was performed by SDS PAGE and Western blotting. PEGylated products can be analyzed by electrophoresis on reducing 6% Tris glycine SDS polyacrylamide gels (Invitrogen). After electrophoresis, the gel can be stained with Coomassie blue to identify all proteins, or standard Western blot procedures can be performed to identify PEGylation patterns on different regions of FVIII. The blots were stained with mouse monoclonal R8B12 or C7F7 antibodies raised against the C-terminal region of the FVIII heavy chain or the N-terminal region of the VIII light chain, respectively, and PEGylation of each chain should be identified. Staining with the 413 antibody directed against the 484-509 region of FVIII will determine whether PEGylation is indeed site-specific for muteins such as PEG1-4. Likewise, staining with a CLB-CAg A antibody that recognizes the 1801-1823 region of FVIII will determine whether PEGylation is indeed site-specific for muteins such as PEG6-10.
显示PEG2(L491C)PEG化对重链的选择性高于轻链，且更具体地，对484-509区域是选择性的(图4)，同时显示PEG6(K1808C)对轻链的选择性高于重链(图5)。PEGylation with PEG2(L491C) was shown to be selective for heavy chains over light chains, and more specifically, for the 484-509 region (Figure 4), while PEG6(K1808C) was shown to be highly selective for light chains in the heavy chain (Figure 5).
对于图4所述的研究，用TCEP还原PEG2突变蛋白(泳道1和8)，随后去除TCEP(泳道2和9)，并用5、12、22、33或43kD PEG-马来酰亚胺处理(泳道3-7和10-14)。未PEG化的FVIII作为未经加工的(H+L)和经加工的重(H)和轻(L)链带运行。所有3条带都在考马斯蓝染色的凝胶上可检测到(右下图)，而用链-特异性的抗体进行的蛋白印迹染色仅仅揭示了未经加工的和对应的链。使用R8B12染色(左上图)，当用PEG-马来酰亚胺处理PEG2时，重链(H)带的强度急剧降低，并产生一条新带，它跑得比与PEG的大小成比例的母H带更高。使用C7F7染色(左下图)，轻链(L)带(由于异质糖基化产生的多条带)不改变强度。两条链的未经加工的H+L带发生移动，因为H链是未经加工的FVIII的部分。考马斯染色也证实了比轻链多得多的重链PEG化，即H带强度的降低。最后，PEG化的带以PEG大小-依赖性的方式在413抗体染色上比R8B12染色损失相对更多的强度(右上图)，推测是由于491的位点特异性的PEG化，这会阻断413抗体与484-509的结合。对于左边的2个凝胶，每个泳道装载的FVIII的量是约30ng，对于右上凝胶，是约1000ng，且对于右下凝胶，是约2000ng。For the studies described in Figure 4, PEG2 muteins were reduced with TCEP (lanes 1 and 8), followed by removal of TCEP (lanes 2 and 9) and treated with 5, 12, 22, 33 or 43 kD PEG-maleimide ( Lanes 3-7 and 10-14). UnPEGylated FVIII operates as unprocessed (H+L) and processed heavy (H) and light (L) chains. All 3 bands were detectable on the Coomassie blue-stained gel (lower right panel), while western blot staining with chain-specific antibodies revealed only unprocessed and corresponding chains. Using R8B12 staining (upper left panel), when PEG2 was treated with PEG-maleimide, the intensity of the heavy chain (H) band was drastically reduced and a new band was generated that ran faster than the parent in proportion to the size of the PEG. The H belt is higher. Using C7F7 staining (lower left panel), the light chain (L) band (multiple bands due to heterogeneous glycosylation) does not change in intensity. The unprocessed H+L bands of the two chains shift because the H chain is part of unprocessed FVIII. Coomassie staining also confirmed much more PEGylation of heavy chains than light chains, ie a reduction in H-band intensity. Finally, the PEGylated band loses relatively more intensity on 413 antibody staining than R8B12 staining in a PEG size-dependent manner (upper right panel), presumably due to site-specific PEGylation of 491, which blocks Binding of the 413 antibody to 484-509. The amount of FVIII loaded per lane was about 30 ng for the left 2 gels, about 1000 ng for the upper right gel, and about 2000 ng for the lower right gel.
还原后去除还原剂，不改变FVIII的迁移(泳道1vs.2和8vs.9)。将22kD PEG加入PEG2阻断413抗体的结合，这与491位置处的特异性的PEG化相一致(图4右上凝胶)。这也暗示着，PEG化的PEG2在人体内具有更低的免疫原性，因为已经显示413抗体具有与人A2抑制性抗体相同的表位(Scandella等，1992，Thromb.Haemost.67，pp.665-71)。Removal of the reducing agent after reduction did not alter the migration of FVIII (lanes 1 vs. 2 and 8 vs. 9). Addition of 22kD PEG to PEG2 blocked binding of the 413 antibody, consistent with specific PEGylation at position 491 (Figure 4 upper right gel). This also implies that PEGylated PEG2 is less immunogenic in humans, since the 413 antibody has been shown to have the same epitope as the human A2 inhibitory antibody (Scandella et al., 1992, Thromb. Haemost. 67, pp. 665-71).
对于图5所述的研究，用TCEP还原PEG6突变蛋白，随后去除TCEP(泳道1和6)，并用5、12、22或33kD PEG-马来酰亚胺处理(泳道2-5和7-10)。未PEG化的FVIII作为未经加工的(H+L)和经加工的重(H)和轻(L)链带运行。因为PEG6(K1808)突变存在于轻链上，所以仅仅在轻链上检测到PEG化，而在重链上未检测到。对于左边的凝胶，每个泳道装载的FVIII的量是约100ng，而对于右边凝胶，是约30ng。For the studies described in Figure 5, PEG6 muteins were reduced with TCEP, followed by removal of TCEP (lanes 1 and 6) and treatment with 5, 12, 22 or 33 kD PEG-maleimide (lanes 2-5 and 7-10 ). UnPEGylated FVIII operates as unprocessed (H+L) and processed heavy (H) and light (L) chains. Since the PEG6(K1808) mutation was present on the light chain, PEGylation was only detected on the light chain but not on the heavy chain. The amount of FVIII loaded per lane was about 100 ng for the gel on the left and about 30 ng for the gel on the right.
在用超过100倍摩尔过量的PEG-马来酰亚胺处理后，甚至在上述的还原和还原剂去除操作后，作为对照运行的BDD没有表现出任何显著的PEG化(图6a)。相同的方法也应用于PEG4和PEG5(图6a)。与PEG2相比，这些突变蛋白未同样有效地PEG化，但是它们对重链的选择性类似于PEG2(L491C)。PEG6(K1808C)PEG化效率相对较低，可能是因为它非常接近N1810处的N-连接的糖基化位点，它会阻断位置1808处的PEG化。因而，我们设计了PEG7(N1810C)来去除1810处的天然糖基化位点。与PEG6相比，在头对头的对比中，PEG7表现出提高的PEG化效率(图6b)。类似地，PEG15表现出比PEG2略微更好的PEG化效率。PEG2+6(BDD的双重突变体)可以在重链和轻链两者上PEG化，因为PEG2是重链半胱氨酸突变，而PEG6是轻链突变(图6c)。该方法也应用于野生型全长FVIII(图6d)。检测PEG化的最大重链片段，其包括A1、A2和大部分B结构域。PEG化模式暗示着单PEG化，且仅存在单个PEG化的半胱氨酸。BDD run as a control did not exhibit any significant PEGylation after treatment with more than a 100-fold molar excess of PEG-maleimide, even after the reduction and reductant removal procedures described above (Fig. 6a). The same approach was also applied to PEG4 and PEG5 (Fig. 6a). These muteins were not as efficiently PEGylated as compared to PEG2, but their selectivity for the heavy chain was similar to PEG2 (L491C). The relatively inefficient PEGylation of PEG6(K1808C) may be due to its close proximity to the N-linked glycosylation site at N1810, which blocks PEGylation at position 1808. Therefore, we designed PEG7(N1810C) to remove the native glycosylation site at 1810. Compared to PEG6, PEG7 exhibited increased PEGylation efficiency in a head-to-head comparison (Fig. 6b). Similarly, PEG15 showed slightly better PEGylation efficiency than PEG2. PEG2+6 (double mutant of BDD) can be PEGylated on both the heavy and light chains since PEG2 is a heavy chain cysteine mutation and PEG6 is a light chain mutation (Fig. 6c). This method was also applied to wild-type full-length FVIII (Fig. 6d). The largest PEGylated heavy chain fragment was detected, which included A1, A2 and most of the B domain. The PEGylation pattern is suggestive of mono-PEGylation, with only a single PEGylated cysteine present.
通过凝血酶切割和蛋白印迹进行PEG化分析。在37℃用凝血酶(40IU/ug FVIII)处理PEG化的产物30分钟。使用的凝血酶也含有APC作为污染物。凝血酶切割将从重链产生50kD A1和43kD A2结构域，而APC切割将A2结构域进一步分离成21和22kD片段(图7)。用识别重链C-末端的R8B12抗体染色，只能鉴别完整的A2结构域和21kD C-末端片段(FVIII562-740)。因而，如果PEG2PEG化是对位置491特异性的，那么43kD A2结构域会被PEG化，但是21kD C-末端片段不会。这确实得到了图7所示的22kD PEG化的PEG2的蛋白印迹的证实。因而，通过消除，PEG2PEG化已经定位在A2结构域的N-末端22kD片段(FVIII 373-561)。因为在pH 6.8时PEG-马来酰亚胺对半胱氨酸是完全选择性的，且仅仅在373-561内的天然FVIII半胱氨酸来自528和554之间的隐蔽二硫键，所以PEG2非常可能在位置491导入的半胱氨酸上PEG化。用FVIII重链N-末端抗体对凝血酶-处理的PEG化的PEG2的蛋白印迹染色显示，A1结构域没有PEG化(未显示数据)。对于5、12、33和43kD的PEG，还已经证实了使用凝血酶切割方法对PEG2的选择性PEG化(未显示数据)。PEG化的野生型全长FVIII的凝血酶切割显示，仅仅B结构域被PEG化(图8)。PEGylation analysis by thrombin cleavage and western blotting. The PEGylated product was treated with thrombin (40 IU/ug FVIII) for 30 minutes at 37°C. The thrombin used also contained APC as a contaminant. Thrombin cleavage will generate 50 kD Al and 43 kD A2 domains from the heavy chain, while APC cleavage further separates the A2 domain into 21 and 22 kD fragments (Figure 7). Staining with the R8B12 antibody recognizing the C-terminus of the heavy chain could only identify the complete A2 domain and the 21 kD C-terminal fragment (FVIII562-740). Thus, if PEG2 PEGylation was specific for position 491, the 43 kD A2 domain would be PEGylated, but the 21 kD C-terminal fragment would not. This was indeed confirmed by the Western blot of 22kD PEGylated PEG2 shown in Figure 7. Thus, by elimination, PEG2 PEGylation has been localized to the N-terminal 22kD fragment of the A2 domain (FVIII 373-561). Because PEG-maleimide is completely selective for cysteines at pH 6.8, and only the native FVIII cysteines within 373-561 come from a cryptic disulfide bond between 528 and 554, PEG2 is most likely PEGylated on the cysteine introduced at position 491. Western blot staining of thrombin-treated PEGylated PEG2 with FVIII heavy chain N-terminal antibody showed no PEGylation of the Al domain (data not shown). Selective PEGylation of PEG2 using the thrombin cleavage method has also been demonstrated for PEGs of 5, 12, 33 and 43 kD (data not shown). Thrombin cleavage of PEGylated wild-type full-length FVIII revealed that only the B domain was PEGylated (Figure 8).
通过碘染色进行PEG化分析。为了证实在考马斯蓝和蛋白印迹染色上新生成的带确实是PEG化的带，使用了钡-碘染色，它对PEG是特异性的(图9)。将PEG化的PEG2在6％Tris甘氨酸凝胶(Invitrogen)上电泳，并用R8B12重链抗体或钡-碘溶液染色(Lee等，Pharm DevTechnol.19994：269-275)。通过使用分子量标记将它们排队，在两种染色之间匹配PEG化的带，从而证实FVIII重链PEG化。PEGylation analysis was performed by iodine staining. To confirm that the newly generated bands on Coomassie blue and Western blot stains were indeed PEGylated bands, barium-iodine staining, which is specific for PEG, was used (Figure 9). PEGylated PEG2 was run on a 6% Tris glycine gel (Invitrogen) and stained with R8B12 heavy chain antibody or barium-iodine solution (Lee et al., Pharm DevTechnol. 19994:269-275). FVIII heavy chain PEGylation was confirmed by matching the PEGylated bands between the two stains by lining them up using molecular weight markers.
通过MALDI-质谱法进行PEG化分析。为了证实重链A2结构域的PEG化，通过基质辅助的激光解吸/离子化(MALDI)质谱法，分析PEG化之前和之后的rFVIII样品。混合样品，并在MALDI靶平板上结晶，其中使用溶于30％乙腈、0.1％TFA中的芥子酸基质。然后在阳性线性模式的Voyager DE-PRO分光计中分析它们。图10显示的结果表明，PEG2的轻链集中在83kD，且重链(HC)集中在89kD。为PEG化的样品获得的光谱显示了HC峰的下降和集中在111kD的新峰的形成。这证实了重链的PEG化。在检出限以上没有观察到PEG化的轻链(在105kD)。PEGylation analysis was performed by MALDI-mass spectrometry. To confirm PEGylation of the heavy chain A2 domain, rFVIII samples before and after PEGylation were analyzed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Samples were pooled and crystallized on MALDI target plates using a sinapinic acid matrix in 30% acetonitrile, 0.1% TFA. They were then analyzed in a Voyager DE-PRO spectrometer in positive linear mode. The results shown in Figure 10 indicate that the light chain of PEG2 is concentrated at 83kD and the heavy chain (HC) at 89kD. The spectrum obtained for the PEGylated sample showed a decrease in the HC peak and the formation of a new peak centered at 111 kD. This confirms PEGylation of the heavy chain. No PEGylated light chain (at 105 kD) was observed above the limit of detection.
然后将两种样品进行凝血酶消化，以20单位凝血酶/mg FVIII，在37℃消化30分钟，随后通过氨基酸分析测定FVIII浓度(Commonwealth Biotechnologies，Inc)。将重链切割成46kD(A1)N-末端级分和43kD(A2)级分。从PEG化的样品(图11)得到的MALDI光谱显示了43kD峰的损失和新的65kD峰的发展，这是由于PEG化的A2结构域。在检出限以上仍然没有观察到LC的PEG化。这些结果再次证实了FVIII的A2结构域的PEG化。将相同的分析应用于PEG化的PEG6，从而证实了轻链A3C1C2片段的PEG化(图12)。Both samples were then subjected to thrombin digestion at 20 units of thrombin/mg FVIII at 37°C for 30 minutes, followed by determination of FVIII concentrations by amino acid analysis (Commonwealth Biotechnologies, Inc). The heavy chain was cleaved into a 46 kD (A1 ) N-terminal fraction and a 43 kD (A2) fraction. The MALDI spectrum obtained from the PEGylated sample (Figure 11) showed the loss of the 43kD peak and the development of a new 65kD peak due to the PEGylated A2 domain. Still no PEGylation of LC was observed above the detection limit. These results again confirm the PEGylation of the A2 domain of FVIII. The same analysis was applied to PEGylated PEG6, confirming PEGylation of the A3C1C2 fragment of the light chain (Figure 12).
活性测量activity measurement
凝结测定。凝固FVIII：C测试方法是基于活化部分凝血激酶时间(aPTT)的一阶段测定。在有因子IXa、钙和磷脂存在下，FVIII起因子X向Xa的酶促转化的辅因子作用。在该测定中，将稀释的测试样品与FVIII缺乏的血浆底物和aPTT试剂的混合物一起在37℃温育。将氯化钙加入温育混合物，并开始凝固。形成凝块所需的时间(秒)和FVIII：C的浓度的对数之间存在反相关。通过对比测试材料的各种稀度的凝固时间和从已知活性的标准材料的系列稀释构建的曲线，内推未知样品的活性水平，并以国际单位/mL(IU/mL)为单位进行报告。Coagulation assay. Clotting The FVIII:C test method is a one-stage assay based on the activated partial thromboplastin time (aPTT). FVIII acts as a cofactor for the enzymatic conversion of Factor X to Xa in the presence of Factor IXa, calcium and phospholipids. In this assay, diluted test samples are incubated at 37°C with a mixture of FVIII-deficient plasma substrate and aPTT reagent. Calcium chloride was added to the incubation mixture and it started to solidify. There was an inverse correlation between the time (seconds) required for clot formation and the logarithm of the concentration of FVIII:C. The activity level of the unknown sample is interpolated and reported in International Units/mL (IU/mL) by comparing the clotting times of various dilutions of the test material to a curve constructed from serial dilutions of a standard material of known activity .
生色测定。生色测定方法由两个连续步骤组成，其中颜色的强度与FVIII活性成比例。在第一步中，在有最佳量的钙离子和磷脂存在下，FIXa和它的辅因子FVIIIa将因子X活化为FXa。存在过量的因子X，从而使得因子X的活化速率仅仅依赖于FVIII的量。在第二步中，因子Xa水解生色底物，以产生生色团，并在405nm光度测定地读出颜色强度。计算未知物的效能，并用斜率比统计学方法检查该测定的有效性。以国际单位/mL(IU/mL)为单位报告活性。Chromogenic assay. The chromogenic assay method consists of two sequential steps in which the intensity of the color is proportional to the FVIII activity. In the first step, FIXa and its cofactor FVIIIa activate factor X to FXa in the presence of optimal amounts of calcium ions and phospholipids. Excess Factor X is present such that the rate of Factor X activation is only dependent on the amount of FVIII. In the second step, Factor Xa hydrolyzes the chromogenic substrate to generate the chromophore, and the color intensity is read photometrically at 405 nm. The power for unknowns was calculated and the validity of the assay was checked using slope ratio statistics. Activity is reported in International Units/mL (IU/mL).
1811-1818环参与与FIX的结合，但是尚未确定该环内单个位置的重要性。相对于天然FVIII，PEG7-10突变蛋白表现出几乎一致的比生色活性(表3)。表3显示了PEG突变蛋白和PEG化的PEG2或PEG6相对于BDD的百分比比活性(S.A.)。通过将生色、凝结或vWF结合活性除以总抗原ELISA(TAE)值，确定S.A.。然后，将PEG化的突变蛋白的S.A.除以BDD的S.A.(8IU/ug生色，5IU/ug凝结，和1vWF/TAE)，并乘以100，以得到表3中列出的百分比S.A.，标题为生色、凝结和vWF/TAE。The 1811-1818 ring is involved in binding to FIX, but the importance of individual positions within this ring has not been determined. The PEG7-10 muteins exhibited almost identical specific chromogenic activity relative to native FVIII (Table 3). Table 3 shows the percent specific activity (S.A.) of PEG muteins and PEGylated PEG2 or PEG6 relative to BDD. S.A. was determined by dividing the chromogenic, clotting or vWF binding activity by the total antigen ELISA (TAE) value. Then, divide the S.A. of the PEGylated mutein by the S.A. of BDD (8 IU/ug chromogenic, 5 IU/ug clotting, and 1 vWF/TAE) and multiply by 100 to obtain the percent S.A. listed in Table 3, titled For chromogenic, condensation and vWF/TAE.
表3.PEG突变蛋白和PEG化的PEG2和PEG6相对于BDD的百分比比活性(S.A.)Table 3. Percent specific activity (S.A.) of PEG muteins and PEGylated PEG2 and PEG6 relative to BDD
如表3中所使用的，“PEG2red”是已经用还原剂处理、然后去除还原剂的PEG2突变蛋白。该还原方法不显著改变FVIII的3种功能活性。缀合到5kD(PEG2-5kD)至43kD(PEG2-43kD)的PEG上的PEG2突变蛋白，不损失显著量的生色活性，但是随着PEG大小增加至超过5kD，具有明显更低的凝结活性。对于更大尺寸的PEG化的PEG2，也存在vWF结合的适度降低。As used in Table 3, "PEG2red" is a PEG2 mutein that has been treated with a reducing agent, followed by removal of the reducing agent. This reduction method does not significantly change the three functional activities of FVIII. PEG2 muteins conjugated to PEG from 5kD (PEG2-5kD) to 43kD (PEG2-43kD) do not lose significant amounts of chromogenic activity, but have significantly lower clotting activity as PEG size increases beyond 5kD . There was also a modest decrease in vWF binding for the larger size PEGylated PEG2.
总抗原ELISA(TAE)。将FVIII捕获到已经用多克隆FVIII抗体包被的微量滴定板上。用生物素化的多克隆rFVIII抗体和链霉抗生物素蛋白辣根过氧化物酶(HRP)缀合物，检测结合的FVIII。过氧化物酶-链霉抗生物素蛋白复合物在加入四甲基联苯胺(TMB)底物后，产生颜色反应。使用4参数拟合模型，从标准曲线内推样品浓度。以μg/mL报告FVIII结果。Total Antigen ELISA (TAE). FVIII was captured onto microtiter plates that had been coated with polyclonal FVIII antibodies. Bound FVIII was detected with a biotinylated polyclonal rFVIII antibody and streptavidin horseradish peroxidase (HRP) conjugate. The peroxidase-streptavidin complex produces a color reaction upon addition of tetramethylbenzidine (TMB) substrate. Sample concentrations were interpolated from the standard curve using a 4 parameter fit model. FVIII results are reported in μg/mL.
vWF结合ELISA。允许FVIII结合严重血友病血浆溶液中的vWf。然后，将FVIII-vWf复合物捕获到已经用vWf-特异性的单克隆抗体包被的微量滴定板上。使用FVIII多克隆抗体和辣根过氧化物酶-抗-兔缀合物，检测结合到vWf上的FVIII。过氧化物酶-缀合的抗体复合物在加入底物后产生颜色反应。使用4参数拟合模型，从标准曲线内推样品浓度。以μg/mL报告FVIII结合结果。PEG化后对任何活性没有显著影响，这与在B结构域的PEG化相一致。vWF binding ELISA. Allows FVIII to bind vWf in severe hemophilia plasma solutions. FVIII-vWf complexes were then captured onto microtiter plates that had been coated with vWf-specific monoclonal antibodies. FVIII bound to vWf was detected using a FVIII polyclonal antibody and a horseradish peroxidase-anti-rabbit conjugate. The peroxidase-conjugated antibody complex produces a color reaction upon addition of the substrate. Sample concentrations were interpolated from the standard curve using a 4 parameter fit model. FVIII binding results are reported in μg/mL. PEGylation had no significant effect on any activity, consistent with PEGylation at the B domain.
表4.用不同大小的PEG进行PEG化之前和之后的野生型全长FVIII(KG-2)的比活性(S.A.)Table 4. Specific activity (S.A.) of wild-type full-length FVIII (KG-2) before and after PEGylation with PEGs of different sizes
通过离子交换层析纯化PEG化的FVIII。将PEG化的FVIII应用于阴离子交换柱或阳离子交换柱，其中蛋白结合到柱上，同时任何过量的游离PEG试剂不结合，并在流通中去除。然后用氯化钠梯度，从柱洗脱PEG突变蛋白。使用钡-碘染色的装载、流通和梯度级分的4-12％Bis-Tris凝胶，证实柱洗脱级分具有PEG化的突变蛋白。PEGylated FVIII was purified by ion exchange chromatography. The PEGylated FVIII is applied to an anion exchange column or a cation exchange column, where the protein is bound to the column while any excess free PEG reagent is not bound and removed in flow-through. The PEG mutein was then eluted from the column using a sodium chloride gradient. Using barium-iodine stained 4-12% Bis-Tris gels of the loading, flow-through and gradient fractions, the column elution fraction was confirmed to have the PEGylated mutein.
通过尺寸排阻层析纯化PEG化的FVIII。合并含有大部分PEG2突变蛋白的阴离子交换级分，并通过超滤浓缩，然后应用于尺寸排阻柱。然后，使用配制缓冲液洗脱柱。因为蛋白大小和形状的差异取决于PEG是否结合在蛋白上，所以该柱可以分离PEG化的PEG2突变蛋白和任何剩余的未PEG化的PEG2。基于具有大部分FVIII活性，合并PEG化的突变蛋白FVIII级分，然后冷冻用于后续动物研究和分子表征。图13对比了未-PEG化的PEG2突变蛋白的洗脱和43kD PEG化的PEG2突变蛋白的洗脱。PEG化的PEG2洗脱明显更早，这指示着由于共价附着的PEG导致的大小和形状的增加。PEGylated FVIII was purified by size exclusion chromatography. Anion exchange fractions containing most of the PEG2 mutein were pooled and concentrated by ultrafiltration before being applied to a size exclusion column. Then, the column was eluted with preparation buffer. Because differences in protein size and shape depend on whether PEG is bound to the protein, this column separates the PEGylated PEG2 mutein from any remaining unPEGylated PEG2. Based on having a majority of FVIII activity, PEGylated mutein FVIII fractions were pooled and then frozen for subsequent animal studies and molecular characterization. Figure 13 compares the elution of the non-PEGylated PEG2 mutein to the elution of the 43kD PEGylated PEG2 mutein. PEGylated PEG2 eluted significantly earlier, indicating an increase in size and shape due to covalently attached PEG.
利用表现出更低PEG化效率(即小于50％)的突变蛋白(如PEG6)，产生高纯度单-PEG化产物的最有效纯化方案是，使用阳离子交换层析和随后的尺寸排阻层析的组合。例如，利用PEG6，阳离子交换层析可以从大多数未-PEG化的PEG6(晚期洗脱级分，图15)中纯化出PEG化的PEG6(早期洗脱级分，图14)。然后，尺寸排阻层析从残存的未-PEG化的蛋白(晚期洗脱级分，图15)中纯化出PEG化的蛋白(早期洗脱级分，图15)。With muteins (such as PEG6) that exhibit lower PEGylation efficiencies (i.e., less than 50%), the most efficient purification scheme to yield high-purity mono-PEGylated products is the use of cation exchange chromatography followed by size exclusion chromatography The combination. For example, using PEG6, cation exchange chromatography can purify PEGylated PEG6 (early eluting fraction, FIG. 14 ) from the majority of non-PEGylated PEG6 (late eluting fraction, FIG. 15 ). Then, size exclusion chromatography purified PEGylated protein (early eluting fraction, FIG. 15 ) from remaining non-PEGylated protein (late eluting fraction, FIG. 15 ).
PEG大小对活性的作用。为了测试PEG大小是否对PEG化后的FVIII的凝结和生色活性有作用，用TCEP还原纯化的全长FVIII、PEG2、PEG6和PEG14，然后去除还原剂，并与缓冲液对照或6kD-64kD的PEG反应。不经去除过量的PEG或未PEG化的FVIII，直接测定得到的PEG化的FVIII。对照实验表明，过量的PEG对FVIII活性无作用。Effect of PEG size on activity. To test whether PEG size has an effect on the coagulation and chromogenic activity of PEGylated FVIII, purified full-length FVIII, PEG2, PEG6, and PEG14 were reduced with TCEP, followed by removal of the reducing agent, and compared with buffer control or 6kD-64kD PEG reaction. The resulting PEGylated FVIII was assayed directly without removal of excess PEG or non-PEGylated FVIII. Control experiments showed that excess PEG had no effect on FVIII activity.
图16显示了该研究的结果。在图16中，将纯化的全长FVIII表达为KG-2。通过将还原并去除还原剂后用PEG处理的样品的值除以用缓冲液对照处理的样品的值，同时考虑PEG化得率，确定图16中报告的百分比活性。对于任何给定的FVIII构建体，所有PEG的PEG化得率是相当的。对于KG-2、PEG2和PEG14，它们是约80％，对于PEG6，是约40％。例如，PEG14缓冲液对照处理的具有6.8IU/mL的凝结活性，相比而言，12kD PEG化的PEG14样品具有3.2IU/mL。但是，PEG化效率是约80％，这意味着3.2IU/mL代表着约80％PEG化和约20％未PEG化的合计活性。假定未PEG化的样品具有与缓冲液对照处理的PEG14相同的活性，PEG化的PEG14的未PEG化的百分比活性计算为34％＝(3.2-6.8乘以20％)/(6.8乘以80％)。Figure 16 shows the results of this study. In Figure 16, purified full-length FVIII was expressed as KG-2. The percent activity reported in Figure 16 was determined by dividing the value of the PEG-treated sample after reduction and removal of the reducing agent by the value of the buffer control-treated sample, taking into account the PEGylation yield. For any given FVIII construct, the PEGylation yields of all PEGs were comparable. For KG-2, PEG2 and PEG14 they are about 80%, for PEG6 they are about 40%. For example, the PEG14 buffer control treated had a clotting activity of 6.8 IU/mL compared to 3.2 IU/mL for the 12kD PEGylated PEG14 sample. However, the PEGylation efficiency was about 80%, which means that 3.2 IU/mL represents a combined activity of about 80% PEGylated and about 20% non-PEGylated. Assuming that the non-PEGylated sample had the same activity as the buffer control treated PEG14, the percent non-PEGylated activity of the PEGylated PEG14 was calculated as 34%=(3.2-6.8 times 20%)/(6.8 times 80% ).
当PEG大小增加到超过6kD时，在BDD的PEG2、PEG6或PEG14位置处A2或A3结构域内的PEG化，导致凝结活性的急剧损失。但是，在全长FVIII的天然B-结构域半胱氨酸处B结构域内的PEG化，对凝结活性无作用。令人感兴趣地，所有PEG化的构建体的生色活性不受影响。这可能是由于测定差异。小生色肽底物可能比在凝结测定中使用的更大蛋白底物更容易接近PEG化的FVIII/FIX/FX复合物。或者，PEG可能影响突变蛋白的激活。一阶段凝结测定可以比二阶段生色测定更容易地检测到这一点。PEGylation within the A2 or A3 domain at the PEG2, PEG6 or PEG14 position of the BDD results in a drastic loss of clotting activity when the PEG size increases beyond 6 kD. However, PEGylation within the B-domain at the native B-domain cysteine of full-length FVIII had no effect on clotting activity. Interestingly, the chromogenic activity of all PEGylated constructs was unaffected. This may be due to assay differences. Small chromogenic peptide substrates may be more accessible to the PEGylated FVIII/FIX/FX complex than larger protein substrates used in clotting assays. Alternatively, PEG may affect the activation of the mutein. A one-stage coagulation assay can detect this more easily than a two-stage chromogenic assay.
为了证实PEG对PEG2、6和14的凝结活性的作用的观察，从过量PEG和未PEG化中纯化出几种PEG化的构建体。由于PEG对生色活性没有任何作用，生色对凝结活性比可以较好地评价PEG对凝结活性的相对作用(表5)。在给定位置(如PEG2)处更大的PEG和更高数目的PEG(如在PEG2+6构建体的情况下)，诱导更大的凝结活性损失。To confirm the observation of the effect of PEG on the coagulation activity of PEG2, 6 and 14, several PEGylated constructs were purified from excess PEG and non-PEGylated. Since PEG has no effect on chromogenic activity, the ratio of chromogenic to coagulation activity can better evaluate the relative effect of PEG on coagulation activity (Table 5). Larger PEGs and higher numbers of PEGs (as in the case of PEG2+6 constructs) at a given position (eg PEG2) induce greater loss of clotting activity.
表5.纯化的PEG化的BDD的生色对凝结比。Table 5. Chromogenic versus coagulation ratios of purified PEGylated BDDs.
＊分支的PEG* Branched PEGs
兔PK研究。为了理解PEG化对FVIII的药物代谢动力学(PK)的作用，在许多物种中进行了PK研究。将NZW SPF兔用于研究：10只雌性，每组5只兔，分成2组(PEG2 FVIII和22kDPEG化的PEG2)。在无菌PBS中稀释样品，终浓度为100IU/mL(生色单位)。每只兔子通过边缘耳静脉接受1ml/kg(100IU/kg)剂量的稀释的测试或对照底物。在注射后的各个时间，在给药后确定的时间点，从中央耳动脉抽取血液样品(1mL)到1mL注射器中(装有100μL 3.8％柠檬酸钠)。将血浆样品与包被在96-孔平板上的R8B12重链抗体一起温育，以特异性地捕获给药的人FVIII。通过生色测定，确定捕获的FVIII的活性(图17)。还将PEG化的PEG2和PEG化的PEG6与BDD相对比(图18和19)，其中PEG化的突变蛋白表现出与BDD相比血浆回收的提高。PEG化的野生型全长FVIII没有表现出很大提高(图20)。Rabbit PK Study. To understand the effect of PEGylation on the pharmacokinetics (PK) of FVIII, PK studies were performed in many species. NZW SPF rabbits were used for the study: 10 females, 5 rabbits per group, divided into 2 groups (PEG2 FVIII and 22kDPEGylated PEG2). Dilute samples in sterile PBS to a final concentration of 100 IU/mL (chromogenic units). Each rabbit received a 1 ml/kg (100 IU/kg) dose of diluted test or control substrate via the marginal ear vein. At various times after injection, blood samples (1 mL) were drawn from the central ear artery into 1 mL syringes (filled with 100 μL of 3.8% sodium citrate) at defined time points after dosing. Plasma samples were incubated with R8B12 heavy chain antibody coated on 96-well plates to specifically capture administered human FVIII. The activity of captured FVIII was determined by chromogenic assay (Figure 17). PEGylated PEG2 and PEGylated PEG6 were also compared to BDD (Figures 18 and 19), where the PEGylated muteins showed improved plasma recovery compared to BDD. PEGylated wild-type full-length FVIII did not show much improvement (Figure 20).
小鼠PK研究。作为第二个物种，在PK研究中使用ICR正常的或血友病的、FVIII缺陷的小鼠(Taconic，Hudson，NY)。在该研究中使用正常小鼠，每个时间点每组5只小鼠。将测试材料稀释进配制缓冲液中，至标称终浓度25IU/mL。通过尾静脉，给每只小鼠施用4mL/kg(约0.1mL总体积)的稀释的测试材料。在指定的时间点，从下腔静脉抽取血液样品(对于正常或血友病小鼠研究，分别是0.45或0.3mL)到1mL注射器中(对于正常或血友病小鼠研究，分别装有50或30μL 3.8％柠檬酸钠)(每份样品一只动物)。使用上述的生色测定方法，测定血浆样品的FVIII浓度。与BDD或PEG6相比，PEG化的PEG6表现出更大的血浆回收(图21)。与BDD相比，PEG化的PEG2表现出更大的血浆回收(图22和23)。Mouse PK study. As a second species, ICR-normal or hemophilic, FVIII-deficient mice (Taconic, Hudson, NY) were used in the PK studies. Normal mice were used in this study, 5 mice per group per time point. Test material was diluted into formulation buffer to a nominal final concentration of 25 IU/mL. 4 mL/kg (approximately 0.1 mL total volume) of the diluted test material was administered to each mouse via the tail vein. At indicated time points, blood samples were drawn from the inferior vena cava (0.45 or 0.3 mL for normal or hemophilic mouse studies, respectively) into 1 mL syringes (50 mL for normal or hemophilic mouse studies, respectively). or 30 μL of 3.8% sodium citrate) (one animal per sample). Plasma samples were assayed for FVIII concentrations using the chromogenic assay described above. PEGylated PEG6 showed greater plasma recovery compared to BDD or PEG6 (Figure 21). PEGylated PEG2 showed greater plasma recovery compared to BDD (Figures 22 and 23).
表6.PEG化的FVIII的PK研究总结，显示了以小时计的血浆半衰期。Table 6. Summary of PK studies of PEGylated FVIII showing plasma half-life in hours.
＊在兔中具有9.6小时半衰期的33kD PEG化的PEG6的初始制品(prep)的纯度不如产生17.4小时的晚期制品。*The initial preparation (prep) of 33 kD PEGylated PEG6 with a half-life of 9.6 hours in rabbits was not as pure as the late preparation resulting in 17.4 hours.
表7.PEG化的PEG突变蛋白在血友病小鼠中的血浆回收。报告了在注射后16小时的血浆回收与在相同时间进行的BDD对照相比的提高倍数。Table 7. Plasma recovery of PEGylated PEG muteins in hemophilic mice. Fold improvements in plasma recovery at 16 hours post injection compared to BDD controls performed at the same time are reported.
血友病小鼠(BDD)因子VIII回收。图24所示的血友病小鼠(BDD)因子VIII回收柱状图描绘了在血友病小鼠测定中两种BDD因子VIII半衰期的药物代谢动力学(PK)评价。该测定设计用于在小鼠模型的静脉内施用后的3个时间点，测量BDD因子VIII(在图24中称作“wt”或野生型BDD因子VIII)和BDD因子VIII的PEG2+6双PEG化变体(在本文其它地方称作BDD因子VIII的L491C，K1808C双变体)的血浆浓度。尽管在0.8和4小时时间点的PK评价是相当的，但16小时评价是特别值得注意的。在16小时，与未-PEG化的分子相比，残存在施用后16小时的小鼠血浆中的双PEG化的BDD因子VIII变体(PEG2+6)的量是约4倍(400％)。Factor VIII recovery in hemophiliac mice (BDD). Figure 24 is a hemophilia mouse (BDD) Factor VIII recovery histogram depicting the pharmacokinetic (PK) evaluation of two BDD Factor VIII half-lives in the hemophilia mouse assay. This assay was designed to measure the PEG2+6 duality of BDD Factor VIII (referred to as "wt" or wild-type BDD Factor VIII in Figure 24) and BDD Factor VIII at three time points after intravenous administration in a mouse model. Plasma concentrations of the PEGylated variant (referred to elsewhere herein as the L491C, K1808C double variant of BDD Factor VIII). Although the PK assessments at the 0.8 and 4 hour time points were comparable, the 16 hour assessment was of particular note. At 16 hours, the amount of double PEGylated BDD Factor VIII variant (PEG2+6) remaining in mouse plasma 16 hours after administration was approximately 4-fold (400%) compared to the non-PEGylated molecule .
肾撕裂伤模型。为了测定PEG化的FVIII突变蛋白是否能有效地终止血友病小鼠的出血，使用了肾撕裂伤模型。用异氟烷麻醉血友病小鼠(具有破坏的FVIII基因的C57/BL6)，并称重。暴露下腔静脉，并使用31号针注射100ul盐水或FVIII。小心地取下针，压迫注射点30-45秒，以防止出血。2分钟后，暴露右肾，并沿着垂直轴将其保持在镊子间。使用15号解剖刀，水平地切入肾3mm深度。为了确保损伤的均匀深度，轻轻将肾保持在中央，以在镊子的每一侧暴露相等的组织。将暴露的肾表面切至镊子的深度。如上所述定量失血。对小鼠测试不同剂量的FVIII，以表征FVIII对肾出血的剂量反应关系。在小鼠肾损伤后，PEG化的PEG2表现出与BDD相当的减少失血的效能(图25)。因而，尽管PEG化的PEG2的凝结活性低于BDD，但该肾撕裂伤模型表明，与BDD相比，PEG化的PEG2的体内效力没有可测量的降低，这与生色测定数据相一致。Renal laceration model. To determine whether PEGylated FVIII muteins can effectively stop bleeding in hemophilic mice, a kidney tear model was used. Hemophilic mice (C57/BL6 with disrupted FVIII gene) were anesthetized with isoflurane and weighed. The inferior vena cava was exposed and 100ul of saline or FVIII was injected using a 31 gauge needle. Carefully remove the needle and apply pressure to the injection site for 30-45 seconds to prevent bleeding. After 2 min, expose the right kidney and hold it between the forceps along the vertical axis. Using a No. 15 scalpel, cut horizontally into the kidney to a depth of 3 mm. To ensure uniform depth of injury, gently hold the kidney in the center to expose equal tissue on each side of the forceps. Cut the exposed kidney surface to the depth of the forceps. Blood loss was quantified as described above. Different doses of FVIII were tested in mice to characterize the dose-response relationship of FVIII on renal hemorrhage. After kidney injury in mice, PEGylated PEG2 exhibited comparable efficacy to BDD in reducing blood loss (Fig. 25). Thus, despite the lower clotting activity of PEGylated PEG2 than BDD, this kidney laceration model showed no measurable reduction in the in vivo potency of PEGylated PEG2 compared to BDD, consistent with the chromogenic assay data.
抗体抑制测定。在位置491特异性地添加高分子量聚合物如聚乙二醇(PEG)(即PEG2)，应降低对mAB 413的结合和敏感性，并扩展至大部分患者抑制性抗体，因为许多患者发展针对相同mAB 413表位的抑制性抗体。为了测试这一点，将递增量的mAB 413与非饱和量(0.003IU/mL)的BDD或43kD PEG化的PEG2一起温育，并在生色测定中测试功能活性(图26)。R8B12(一种非抑制性抗体)和ESH4(一种靶向C2结构域的抑制性抗体)用作对照。PEG化的PEG2对mAB 413抑制的抗性确实超过对BDD，并在有不结合491位置附近的对照抗体存在下，表现出类似的抑制模式。此外，PEG抗mAB 413抑制的保护作用依赖于PEG大小，其中更大的PEG具有更大的作用(图27)。为了测试PEG化的FVIII是否更抗来自患者的抑制剂抗体，在有一组源自血友病A患者(它们已经发展了对FVIII的抑制剂)的血浆存在下，测量生色活性。在测试的8份患者血浆中，43kD PEG化的PEG2对患者血浆抑制的抗性比4份患者血浆样品中的BDD的更高。例如，PEG化的PEG2、PEG6或PEG2+6在一份患者血浆中表现出比BDD更大的残余活性，但是在另一份血浆中并非如此(图28)。双PEG化的PEG2+6似乎比单PEG化的PEG2或PEG6的抗性更高。这些结果表明，PEG化的PEG突变蛋白可以更有效地治疗已经发展对FVIII的抑制剂的患者。Antibody Inhibition Assay. Specific addition of a high molecular weight polymer such as polyethylene glycol (PEG) at position 491 (i.e., PEG2) should reduce binding and sensitivity to mAB 413 and extend to the majority of patients with inhibitory antibodies, as many develop antibodies against Inhibitory antibodies to the same mAB 413 epitope. To test this, increasing amounts of mAB 413 were incubated with non-saturating amounts (0.003 IU/mL) of BDD or 43kD PEGylated PEG2 and tested for functional activity in a chromogenic assay (Figure 26). R8B12 (a non-inhibitory antibody) and ESH4 (an inhibitory antibody targeting the C2 domain) were used as controls. PEGylated PEG2 was indeed more resistant to inhibition by mAB 413 than BDD and showed a similar pattern of inhibition in the presence of a control antibody that did not bind near the 491 position. Furthermore, the protective effect of PEG against mAB 413 inhibition was dependent on PEG size, with larger PEG having a greater effect (Figure 27). To test whether PEGylated FVIII is more resistant to inhibitor antibodies from patients, chromogenic activity was measured in the presence of plasma from a panel of hemophilia A patients who had developed inhibitors to FVIII. Of the 8 patient plasmas tested, the 43kD PEGylated PEG2 was more resistant to patient plasma inhibition than BDD in the 4 patient plasma samples. For example, PEGylated PEG2, PEG6 or PEG2+6 showed greater residual activity than BDD in one patient plasma but not in another (Figure 28). Double PEGylated PEG2+6 appeared to be more resistant than single PEGylated PEG2 or PEG6. These results suggest that PEGylated PEG muteins may be more effective in treating patients who have developed inhibitors to FVIII.
高通量PEG化筛选。特定PEG突变蛋白的PEG化效率是不可预测的，特别是由于没有BDD的直接结构信息。例如，基于BDD的结构模型，人们可以预测PEG4和PEG5的PEG化效率非常高，类似于PEG2和PEG15的PEG化效率，因为根据结构，所有3个位置都是表面暴露的，且朝向外侧。因而，为了使用PEG通过系统的PEG化来搜索新的清除机理，需要筛选大量的突变蛋白。High-throughput PEGylation screening. The PEGylation efficiency of a specific PEG mutein is unpredictable, especially since there is no direct structural information on the BDD. For example, based on the structural model of BDD, one can predict that the PEGylation efficiency of PEG4 and PEG5 is very high, similar to that of PEG2 and PEG15, because according to the structure, all 3 positions are surface exposed and towards the outside. Thus, in order to search for new clearance mechanisms by systematic PEGylation using PEG, a large number of mutant proteins needs to be screened.
为了快速地筛选大量的PEG突变蛋白，已经开发了新的高通量方法，其可以测试来自瞬时转染的突变蛋白的PEG化产物的PEG化效率和功能活性。使用Amicon-centra Ultra装置MWCO 30K，将少至5-10mL的具有低至0.1-0.2IU/mL的FVIII生色值的瞬时表达的PEG突变蛋白浓缩约50倍，从而使得FVIII的浓度达到1nM以上，接近抗体与FVIII相互作用的亲和范围。将浓缩的PEG突变蛋白(约300uL)与约30uL的C7F7FVIII抗体树脂一起在4℃温育过夜，洗涤，洗脱，渗析，并还原。去除还原剂，PEG化还原的PEG突变蛋白，在蛋白印迹上运行，如上所述(图29和30)。瞬时表达的PEG突变蛋白的相对PEG化效率准确地匹配纯化的PEG突变蛋白。To rapidly screen large numbers of PEG muteins, new high-throughput methods have been developed that can test PEGylation efficiency and functional activity of PEGylated products from transiently transfected muteins. Using an Amicon-centra Ultra device MWCO 30K, as little as 5-10 mL of transiently expressed PEG mutein with FVIII chromogenic values as low as 0.1-0.2 IU/mL was concentrated approximately 50-fold, resulting in a concentration of FVIII above 1 nM , close to the affinity range where the antibody interacts with FVIII. The concentrated PEG mutein (about 300 uL) was incubated with about 30 uL of C7F7FVIII antibody resin overnight at 4°C, washed, eluted, dialyzed, and reduced. The reducing agent was removed, and the reduced PEG muteins were PEGylated and run on Western blots as described above (Figures 29 and 30). The relative PEGylation efficiency of the transiently expressed PEG muteins exactly matched that of the purified PEG muteins.
通过该方法，在1-2个月内，可以筛选许多PEG突变蛋白。例如，PEG14(K1804C BDD)具有至少约80％用12kD PEG对轻链的PEG化，而没有PEG化的重链(未显示数据)，这与位于轻链上的K1804C突变相一致。基于BDD结构，K1804和K1808之间的Cα至Cα距离(PEG6位置)仅是8.4埃，从而表明在该位置导入43kD PEG具有与33kD PEG化的PEG6相似的PK提高，优点是高得多的PEG化得率。在表8中总结了测试的所有PEG突变蛋白的相对PEG化得率。PEG化对其中导入了半胱氨酸突变的特定FVIII链是高度选择性的，因为每个在重链中具有半胱氨酸的突变蛋白仅在重链上被PEG化，而每个在轻链中具有半胱氨酸的突变蛋白仅在轻链上被PEG化。突变蛋白号2-31代表着BDD的半胱氨酸突变，其用半胱氨酸替代在列出的位置处的天然氨基酸。PEG2+6是BDD的双突变蛋白，其中位置491和1808被半胱氨酸替代。A1和A2(和KG-2(即全长FVIII)的B结构域)属于重链，而A3、C1和C2属于轻链。通过在SDS PAGE上电泳PEG化的产物，对比PEG化的带和未PEG化的带的强度，估计PEG化效率：+++约＞80％PEG化得率，++约30-70％得率，+约10-30％得率，-约＜10％得率。By this method, within 1-2 months, many PEG muteins can be screened. For example, PEG14 (K1804C BDD) had at least about 80% PEGylation of the light chain with 12kD PEG, but no PEGylated heavy chain (data not shown), consistent with the K1804C mutation located on the light chain. Based on the BDD structure, the Cα to Cα distance (PEG6 position) between K1804 and K1808 is only 8.4 angstroms, thus suggesting that introduction of 43kD PEG at this position has a similar PK improvement as 33kD PEGylated PEG6, with the advantage of much higher PEG Yield. The relative PEGylation yields of all PEG muteins tested are summarized in Table 8. PEGylation is highly selective for specific FVIII chains into which cysteine mutations have been introduced, since each mutein with a cysteine in the heavy chain is PEGylated only on the heavy chain, whereas each mutant protein on the light chain Muteins with cysteines in the chain were PEGylated only on the light chain. Mutant protein numbers 2-31 represent cysteine mutations of BDD that substitute cysteine for the natural amino acid at the positions listed. PEG2+6 is a double mutein of BDD in which positions 491 and 1808 are replaced by cysteines. A1 and A2 (and the B domain of KG-2 (ie, full-length FVIII)) belong to the heavy chain, while A3, C1 and C2 belong to the light chain. By electrophoresis of PEGylated products on SDS PAGE, comparing the intensity of PEGylated bands and non-PEGylated bands, the PEGylation efficiency is estimated: +++ about >80% PEGylation yield, ++ about 30-70% Yield, + about 10-30% yield, - about <10% yield.
表8各种PEG化的FVIII的PEG化效率Table 8 PEGylation efficiency of various PEGylated FVIII
还原的PEG突变蛋白的质谱法分析。为了确定预防PEG突变蛋白或全长FVIII的直接PEG化的“帽”的特性，用浓度范围为67uM-670uM的TCEP还原PEG2+14。PEG化得率与递增量的TCEP成比例地增加(图31)。在PEG化之前，还通过质谱法分析相同的样品(图32)。为了得到可以直接研究的蛋白结构域，在37℃，用比率为20单位/mg FVIII的凝血酶消化样品30分钟。凝血酶切割产生A2片段，其包括残基372-740，且没有占据的糖基化位点。将消化的样品注射到C4反相液相层析系统上，且通过电喷射界面，将来自柱的洗脱液直接导入四极飞行时间质谱仪中。解卷积与A2结构域相对应的层析峰以下的质谱，以提供蛋白的完整质量值。在还原之前，PEG2+14的A2结构域产生比理论预测大118道尔顿的质量。随着TCEP浓度的增加，出现一个新峰，它具有A2结构域的准确预测质量。该新峰的比例随着TCEP浓度的增加而增加。118道尔顿差异可以归因于在残基Cys 491处与半胱氨酸(119Da)形成二硫键的半胱氨酰化和仪器准确度。因而，这表明，PEG突变蛋白可以被半胱氨酸加帽，这会阻止直接的PEG化。Mass spectrometry analysis of reduced PEG muteins. To determine the properties of the "cap" that prevents direct PEGylation of PEG muteins or full-length FVIII, PEG2+14 was reduced with TCEP at concentrations ranging from 67uM-670uM. The yield of PEGylation increased proportionally with increasing amounts of TCEP (Figure 31). The same samples were also analyzed by mass spectrometry prior to PEGylation (Figure 32). To obtain protein domains that can be directly studied, samples were digested with thrombin at a rate of 20 units/mg FVIII for 30 minutes at 37°C. Thrombin cleavage yields the A2 fragment, which includes residues 372-740, and has no occupied glycosylation sites. Digested samples were injected onto a C4 reverse phase liquid chromatography system and the eluate from the column was directed into a quadrupole time-of-flight mass spectrometer via an electrospray interface. The mass spectrum below the chromatographic peak corresponding to the A2 domain was deconvoluted to provide the full mass value of the protein. Before reduction, the A2 domain of PEG2+14 yielded a mass 118 Daltons larger than theoretically predicted. As the concentration of TCEP increased, a new peak appeared with the accurately predicted mass of the A2 domain. The proportion of this new peak increases with increasing TCEP concentration. The 118 Dalton difference can be attributed to cysteinylation and instrument accuracy to form a disulfide bond with cysteine (119 Da) at residue Cys 491. Thus, this suggests that PEG muteins can be capped with cysteines, which would prevent direct PEGylation.
本文公开的所有文献都在本文中整体引作参考。All documents disclosed herein are incorporated herein by reference in their entirety.
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